Compositions and Methods for Inducing Angiogenesis

ABSTRACT

An isolated peptide comprising an amino acid sequence HWRR as set forth by SEQ ID NO:5, the peptide consists of 4 or 5 amino acids, is provided. Also provided are methods of treating angiogenesis-related pathologies using the peptide of the invention or pharmaceutical compositions comprising same.

FIELD AND BACKGROUND OF THE INVENTION

Angiogenesis is the process of generating new capillary blood vesselsand involves an interplay between cells and soluble factors. Thus,activated endothelial cells migrate and proliferate to form new vessels,which are surrounded by layers of periendothelial cells; small bloodvessels are surrounded by pericytes and large blood vessels aresurrounded by smooth muscle cells.

Numerous factors are known to regulate angiogenesis. These includesoluble factors and tissue oxygen. Factors known to positively regulateangiogenesis include Vascular Endothelium Growth Factor (VEGF), basicFibroblast Growth Factor (bFGF), acidic FGF/FGF-1 and hypoxia-induciblefactor-1α (HIF-1α). Hypoxia induces the expression of several geneproducts such as erytropoietin, VEGF, bFGF and glycolytic enzymes.

Angiogenesis-dependent pathologies result from disregulatedangiogenesis, i.e., excessive amounts of new blood vessels orinsufficient number of blood vessels. Insufficient angiogenesis isrelated to a large number of diseases and conditions, such as coronaryartery diseases, delayed wound healing, delayed ulcer healing,reproduction associated disorders, arteriosclerosis, myocardialischemia, peripheral ischemia, cerebral ischemia, retinopathy,remodeling disorder, von Hippel-Lindau syndrome, diabetes and hereditaryhemorrhagic telengiectasia. On the other hand, excess of angiogenesis ischaracteristic to cancerous cells and cancer metastasis.

Common treatment of ischemic diseases (e.g., peripheral artery diseasessuch as critical limb ischemia, coronary artery disease) involvesmechanical revascularization by percutaneous techniques or a bypasssurgery using arterial and venous conduits as grafts onto the coronaryarterial tree. However, these treatment modalities have significantlimitations in individuals with diffuse atherosclerotic disease orsevere small vessel coronary artery disease, in diabetic patients, aswell as in individuals who have already undergone surgical orpercutaneous procedures. For these reasons, therapeutic angiogenesis,aimed at stimulating new blood vessel growth, is highly desirable.

The therapeutic concept of angiogenesis therapy is based on the premisethat the existing potential for vascular growth inherent to vasculartissue can be utilized to induce the development of new blood vesselsunder the influence of the appropriate angiogenic molecules.

Animal studies have proven the feasibility of enhancing collateralperfusion and function in experimental models of acute and chronicischemia via exogenous angiogenic compounds (Sun Q, Chen R R, Shen Y,Mooney D J, Rajagopalan S, Grossman P M. Sustained vascular endothelialgrowth factor delivery enhances angiogenesis and perfusion in ischemichind limb. Pharm. Res. 2005; 22, 1110-6). In addition, syntheticpeptides encompassing portions of the human FGF and VEGF proteins weredescribed to efficiently agonize or antagonize the biological functionsof the growth factor family members. Furthermore, screening acombinatorial phage display library of random 12-mer peptides resultedin isolation of specific peptides capable of binding the cell-surface ofendothelial cells and triggering angiogenic processes which includedendothelial cell-proliferation and vascularization (PCT Pub.WO2005/039616 to the present inventors).

Members of the ADAM (A Disintegrin And Metalloproteinase) family ofproteolytic enzymes are implicated in the processing of many singletransmembrane-bound proteins ranging from cell surface receptors togrowth factors and cytokines. The disintegrin domains in the ADAMproteins compete with extracellular proteins (ECM) on integrin binding.As such, the ADAM proteins are thought to be involved in the regulationof cell/ECM- and cell/cell-interactions in many physiological andpathophysiological conditions. In addition, the conservedmetalloprotease domain in ADAM proteins is thought to be involved inshedding of biologically important cell surface proteins. Thus, it wassuggested that ADAM proteases could facilitate cell migration byshedding ectodomains and by remodeling of the ECM (Trochon V., et al.,1998).

ADAM15 (also known as metargidin) is a membrane-anchored glycoproteinimplicated in cell-cell or cell-matrix interactions and in theproteolysis of molecules on the cell surface or extracellular matrix.The expression level of ADAM15 was found to be elevated in numeroustissues and conditions characterized by extensive remodeling such asvascular cells, endocardium, atherosclerotic lesions, rheumatoid tissue,chondrosarcoma and atrial fibrillation and dilatation. In addition,ADAM15 is expressed in human aortic smooth muscle and cultured UmbilicalVein Endothelial Cells (HUVECs) and the ADAM15 gene was localized tohuman chromosome band 1q21.3 that is amplified in several types ofcancers.

The possible role of ADAM15 in neovascularization was studied in micelacking the ADAM15 gene (i.e., ADAM15 knock out mice). The ADAM15 knockout mice exhibit a major reduction in neovascularization compared towild-type controls (Bohm B B, Aigner T, Roy B Brodie T A, Blobel C PBurkhardt H; Arthritis Rheum. 2005 52, 4 1100-9); a strongly reducedangiogenic response in a model of hypoxia-induced proliferativeretinopathy; and significantly smaller tumors which develop afterimplantation of melanoma cells. Specific candidate substrates for ADAM15in the context of neovascularization include Notchl and -4, PECAM-1,VE-cadherin, TIE-2, membrane type 1 MMP and possibly also Kit-ligand. Onthe other hand, although ADAM15 demonstrates strong and specificinteractions with hematopoietic Src family kinases, which are known tobe required for VEGF-mediated angiogenesis, nor VEGF or bFGF inducechanges in ADAM15 expression in HUVECs.

Inhibition of ADAM15 by specific antibodies or the metalloproteaseinhibitor BB3103 resulted in blockage of human mesangial cell migrationand suggested that the metalloprotease activity is essential for thisprocess.

The glucose-regulated protein (GRP78) (also known as HSPA5 or BiP), is amember of the heat-shock protein-70 (HSP70) family, highly conservedmolecules that act as molecular chaperones and is involved in thefolding and assembly of proteins in the endoplasmic reticulum (ER).GRP78 was found to be upregulated in drug-resistant lung cancer celllines and its expression level was inversely correlated to themicrovessel density (MVD) [Koomaqi R., et al., 1999; Anticancer Res.19(5B): 4333-6]. In contrast, inhibition of GRP78 using smallinterfering RNA resulted in sensitization of human breast cancer cellsto etoposide-mediated cell death [Dong D., et al., 2005, Cancer Res.65(13): 5785-91]. On the other hand, no correlation was found betweenthe expression level of ER-stress response protein GRP78 and theresistance to hypoxia or ER stresses [Koshikawa N., et al., 2006,Oncogene 25(6):917-28]. Recently, GRP78 was found to be exposed on thecell surface of proliferating endothelial cells and stressed tumor cellsand to play a key role in the anti-angiogenic and antitumor activity ofKringle 5 (K5) [Davidson D J., et al., 2005, Cancer Res. 65(11):4663-72].

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated peptide comprising an amino acid sequence HWRR as set forth bySEQ ID NO:5, wherein the peptide consists of 4 or 5 amino acids.

According to another aspect of the present invention there is providedan isolated peptide comprising an amino acid sequence HWRR as set forthby SEQ ID NO:5, with the proviso that the peptide is not SEQ ID NO:11(YPHIDSLGHWRR).

According to yet another aspect of the present invention there isprovided a composition-of-matter comprising at least one peptide of theinvention.

According to still another aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientat least one peptide of the invention and a pharmaceutically acceptablecarrier or diluent.

According to an additional aspect of the present invention there isprovided a method of inducing angiogenesis in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of at least one peptide of the invention, to thereby induceangiogenesis in the subject.

According to yet an additional aspect of the present invention there isprovided use of at least one peptide of the invention for themanufacturing of a medicament identified for inducing angiogenesis in atissue of a subject.

According to still an additional aspect of the present invention thereis provided use of at least one peptide of the invention for themanufacturing of a medicament identified for treating a pathologyselected from the group consisting of delayed wound-healing, delayedulcer healing, reproduction associated disorder, arteriosclerosis,ischemic vascular disease, ischemic heart disease, myocardial ischemia,myocardial infarction, heart failure, myocardial dysfunction, myocardialremodeling, cardiomyopathies, coronary artery disease (CAD),atherosclerotic cardiovascular disease, left main coronary arterydisease, arterial occlusive disease, peripheral ischemia, peripheralvascular disease, vascular disease of the kidney, peripheral arterialdisease, limb ischemia, critical leg ischemia, lower extremity ischemia,cerebral ischemia, cerebro vascular disease, retinopathy, retinalrepair, remodeling disorder, von Hippel-Lindau syndrome, diabetes,hereditary hemorrhagic telengiectasia, ischemic vascular disease,Buerger's disease and ischemia associated with neurodegenerative diseasesuch as Parkinson's and Alzheimer's disease.

According to a further aspect of the present invention there is provideda method of treating a pathology characterized by insufficientangiogenesis in a tissue of a subject, the method comprisingadministering to the subject a therapeutically effective amount of atleast one peptide of the invention, to thereby treat the pathologycharacterized by insufficient angiogenesis in the tissue of the subject.

According to yet a further aspect of the present invention there isprovided use of at least one peptide of the invention for themanufacturing of a medicament identified for treating a pathologycharacterized by insufficient angiogenesis in a tissue of a subject.

According to still a further aspect of the present invention there isprovided a composition for targeting an agent to endothelial cells, thecomposition comprising the agent attached to a peptide of the invention.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe composition of the invention and a pharmaceutically acceptablecarrier or diluent.

According to still a further aspect of the present invention there isprovided a method of treating a pathology characterized by abnormallyincreased angiogenesis, comprising administering to a subject in needthereof a therapeutically effective amount of the composition of theinvention, thereby treating the pathology characterized by theabnormally increased angiogenesis.

According to still a further aspect of the present invention there isprovided use of the composition of the invention for the manufacturingof a medicament identified for the treatment of a pathologycharacterized by abnormally increased angiogenesis.

According to still a further aspect of the present invention there isprovided a method of identifying a putative angiogenic molecule, themethod comprising: (a) providing endothelial cells having the peptide ofthe invention bound thereto, and (b) identifying a molecule capable ofdisplacing the peptide from the endothelial cells, to thereby identify aputative angiogenic molecule.

According to still a further aspect of the present invention there isprovided a method of identifying a putative angiogenic molecule, themethod comprising: (a) incubating the peptide of the invention with aglucose-regulated protein (GRP78) or cells expressing the GRP78 underconditions suitable for formation of a complex between the peptide andthe GRP78 or the cells expressing GRP78, and (b) identifying a moleculecapable of displacing the peptide from the complex, to thereby identifya putative angiogenic molecule.

According to further features in the embodiments of the inventiondescribed below, the amino acid sequence is HWRRP (SEQ ID NO:7) or HWRRA(SEQ ID NO:8).

According to still further features in the described embodiments theamino acid sequence is set forth by SEQ ID NO:2.

According to still further features in the described embodiments theamino acid sequence is set forth by SEQ ID NO:3.

According to still further features in the described embodiments theamino acid sequence is set forth by SEQ ID NO:4.

According to still further features in the described embodiments thepeptide is a linear peptide.

According to still further features in the described embodiments thepeptide is a cyclic peptide.

According to still further features in the described embodiments thepeptide consists of 12 or less amino acids.

According to still further features in the described embodiments thepathology characterized by insufficient angiogenesis in the tissue ofthe subject is selected from the group consisting of delayedwound-healing, delayed ulcer healing, reproduction associated disorder,arteriosclerosis, ischemic vascular disease, ischemic heart disease,myocardial ischemia, myocardial infarction, heart failure, myocardialdysfunction, myocardial remodeling, cardiomyopathies, coronary arterydisease (CAD), atherosclerotic cardiovascular disease, left maincoronary artery disease, arterial occlusive disease, peripheralischemia, peripheral vascular disease, vascular disease of the kidney,peripheral arterial disease, limb ischemia, critical leg ischemia, lowerextremity ischemia, cerebral ischemia, cerebro vascular disease,retinopathy, retinal repair, remodeling disorder, von Hippel-Lindausyndrome, diabetes, hereditary hemorrhagic telengiectasia, ischemicvascular disease, Buerger's disease and ischemia associated withneurodegenerative disease such as Parkinson's and Alzheimer's disease.

According to still further features in the described embodiments themethod or the use of the invention is for treating a pathology selectedfrom the group consisting of delayed wound-healing, delayed ulcerhealing, reproduction associated disorder, arteriosclerosis, ischemicvascular disease, ischemic heart disease, myocardial ischemia,myocardial infarction, heart failure, myocardial dysfunction, myocardialremodeling, cardiomyopathies, coronary artery disease (CAD),atherosclerotic cardiovascular disease, left main coronary arterydisease, arterial occlusive disease, peripheral ischemia, peripheralvascular disease, vascular disease of the kidney, peripheral arterialdisease, limb ischemia, critical leg ischemia, lower extremity ischemia,cerebral ischemia, cerebro vascular disease, retinopathy, retinalrepair, remodeling disorder, von Hippel-Lindau syndrome, diabetes,hereditary hemorrhagic telengiectasia, ischemic vascular disease,Buerger's disease and ischemia associated with neurodegenerative diseasesuch as Parkinson's and Alzheimer's disease.

According to still further features in the described embodiments thepeptide is capable of binding a glucose-regulated protein (GRP78) as setforth by SEQ ID NO:9 on endothelial cells of the tissue.

According to still further features in the described embodiments thebinding of the peptide to the glucose-regulated protein (GRP78) as setforth by SEQ ID NO:9 is capable of inducing angiogenesis.

According to still further features in the described embodiments theagent is selected from the group consisting of a toxin, achemotherapeutic agent and a radioisotope.

According to still further features in the described embodiments thepathology is selected from the group consisting of cancer, metastaticcancer, myelodysplasia, Systemic mastocytosis (SM), retinalneovascularization, neovascularization in atherosclerotic plaques,hemangiomas, arthritis and psoriasis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. In case of conflict, the patent specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereof.This term encompasses the terms “consisting of” and “consistingessentially of”.

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the biotechnology art.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of theembodiments of the invention only, and are presented in the cause ofproviding what is believed to be the most useful and readily understooddescription of the principles and conceptual aspects of the invention.In this regard, no attempt is made to show structural details of theinvention in more detail than is necessary for a fundamentalunderstanding of the invention, the description taken with the drawingsmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic illustration depicting the domain structure ofADAM family members which is represented by the ADAM15 molecule. ADAMproteins typically contain a pro domain (PRO), a metalloprotease domain(MP) and disintegrin domains (D) which include the tripeptide RGD motifthat is also found in extracellular matrix (ECM) proteins and whichdetermines specificity and affinity of the ADAMs to distinct integrins.The ADAM proteins also include cysteine rich (C) region, an EGF likedomain and a transmembrane segment (TM, in black), along with acytoplasmic tail (tail). Upon removal of the pro domain, themetalloproteinase domain is activated. The Pro domain keeps the enzymein an inactive state. Pro domain removal occurs either through theaction of pro-hormone convertases or by autocatalysis.

FIGS. 2 a-b depict the ADAM15 protein domains (FIG. 2 a) and amino acidsequence (FIG. 2 b). FIG. 2 a—the amino acid positions of the ADAM15protein domains refer to the polypeptide set forth by SEQ ID NO:1(GenBank Accession No. Q13444); FIG. 2 b—amino acid sequence of theADAM15 protein. Bolded text refers to the metalloprotease domain,disintegrin like domain and EGF like domain. The underlined textcorresponding to amino acids 286-297 refers to the ADOPep1 peptide beingonly in the metaloprotease domain but not in the disintegrin like domainor the EGF like domain.

FIG. 3 is a FACS analysis depicting the binding (in percentages) ofbiotinylated ADOPep1 (ADOPep1^(Biot)), an ADAM15 derived peptide (SEQ IDNO:2), to endothelial cells (EC) under normoxia. Biotinylated ADOPep1 at0.05, 0.5 and 5 microgram/ml (μg/ml) were added to endothelial cells.Note the increase in binding of ADOPep1 to endothelial cells whichreached about 62% in a dose dependent manner and was maximal at 5 μg/ml.

FIG. 4 is a FACS analysis depicting the binding (in total counts) ofBiotinylated ADOPep1 to endothelial cells under normoxia and hypoxiaconditions. Note that the binding of ADOPep1 added at 5 micrograms per100,000 cells to endothelial cells increased under hypoxia. Xaxis—Intensity of binding.

FIG. 5 is a graph depicting cell proliferation as a function of ADOPeps'concentration. Proliferation of endothelial cells (EC) under hypoxiaconditions was measured by [H³]Thymidine incorporation. Endothelialcells (12,000/well) in minimal growth medium were incubated for 24 hoursin the presence of 1, 10 and 100 ng/ml of ADOPep1 (SEQ ID NO:2; reddiamonds), 2 (SEQ ID NO:3; blue squares) and 3 (SEQ ID NO:4; greentriangles) peptides. Note that ADOpep1 induced proliferation ofendothelial cells under hypoxia conditions at a concentration of 10ng/ml.

FIG. 6 is a graph depicting migration of endothelial cells under hypoxiaconditions. Endothelial cells (25,000) were incubated in endothelialcells growth medium free supplements in the migration chamber. ADOPep1(SEQ ID NO:2), 2 (SEQ ID NO:3) and 3 (SEQ ID NO:4) were added to thefeeder tray of the migratory kit at 1, 10 and 100 ng/ml for 5 hourshypoxia. Results were determined by a fluorescence ELISA reader andexpressed as increase in percentage migration in Relative fluorescenceUnits. Note that ADOPep 1 induced increase in percent of migration at 10ng/ml.

FIG. 7 is a graph depicting blood perfusion determined in a mouseischemic hind limb model by laser Doppler blood flow analyzer anddemonstrating the in vivo activity of ADOPeps on restoration of bloodperfusion in ischemic mouse hind limb. A mouse hind limb model wascreated by excision of femoral artery of C57B1 mouse hind limb, thenon-operated limb served as a control. ADOPep1 (SEQ ID NO:2; redsymbols) and 3 (SEQ ID NO:4; green symbols) were injectedintra-muscularly one day after surgery at 0.1 (squares) and 1 (circles)micrograms/mouse. Injection of PBS (black diamond) was used as acontrol. Blood flow was measured using a laser Doppler immediately (TO)or at 7 (T7), 14 (T14) and 21 (T21) days after surgery. The averageperfusion of each limb was determined and the perfusion ratio [expressedas Relative Perfusion (ischemic left/control right leg)] was plottedagainst time. Note the significant increase in blood perfusion (p<0.05)in limb of mice treated with ADOPep1 at 0.1 microgram on day 21 aftersurgery.

FIG. 8 is a graph depicting histological assessment of angiogenesis inthe ischemic hind limb treated with ADOPep1. Mice were subjected to hindlimb ligation and ADOPep administration as described in FIG. 7 and atthe noted days (0, 7, 14 and 21 days) post hind limb ligation the micewere sacrificed and legs were embedded in paraffin. Sections werestained with anti von Willebrand factor and the number of Factor VIIIpositive vessels was determined using the Image Pro Plus software. Shownare the average blood vessel densities (of 10 individual microscopicalfields per sample) expressed as number of capillaries per millimetersquared in histological sections of mouse hind limb ischemia treatedwith ADOPep1. Note the significant increase in the blood vessel densityof ischemic hind limb following injection of the ADOPep1.

FIGS. 9 a-b are microscopical images of histological sections subjectedto von Willebrand Factor immuno-staining of mouse hind limb ischemia 21days following injection of ADOPep1 (FIG. 9 a) or PBS (FIG. 9 b). Notethe significantly higher von Willebrand Factor staining of small vesselsin the ADOPep1 treated group (FIG. 9 a) as compared to the control (FIG.9 b). Magnification x200.

FIGS. 10 a-b are images of Coomassie blue staining of polyacrylamide gelelecrophoresis (PAGE) of endothelial cell lysates obtained from cellsunder hypoxia and analyzed before (FIG. 10 a) and after (FIG. 10 b)immunoprecipitation (IP) of the cells with biotinylated ADOPep1. FIG. 10a—lane 1—endothelial cell lysate before IP; lane 2—molecular weightmarkers; FIG. 10 b—lane 3—IP endothelial cell lysate with ADOPep1. Notethe major single protein band of 78 kDa following IP with biotinylatedADOPep1.

FIG. 11 is a Western Blot analysis of immune precipitation ofendothelial cells lysate with biotinylated ADOPep1 under normoxia andhypoxia conditions. Staining of the nitrocellulose membrane withbiotinylated ADOPep1 followed by Chemiluminescent Substrate revealed aband at 78 kDa which was further identified by Mass specrometry as GRP78protein (GenBank Accession No. CAB71335; SEQ ID NO:9). Lane 1—IP ofendothelial cells lysate under normoxia with biotinylated ADOPep1; lane2—IP of endothelial cells lysate under hypoxia with biotinylatedADOPep1.

FIG. 12 is Western Blot analysis of immune precipitation of endothelialcells lysate under hypoxia and normoxia with biotinylated ADOPep1.Staining of the nitrocellulose membrane with anti GRP78 antibody (SantaCruz Biotechnologies, CA, USA) followed by Chemiluminescent Substrateconfirmed the identity of the GRP78 protein in the major 78 kDa proteinband. Lane 1—IP of endothelial cells lysate under hypoxia withbiotinylated ADOPep1; lane 2—IP of endothelial cells lysate undernormoxia with biotinylated ADOPep1.

FIGS. 13 a-b are Western blot analyses of immune precipitation ofendothelial cells lysate under hypoxia with biotinylated ADOPep1.Staining of the nitrocellulose membrane was performed with BiotinylatedADOPep1 (FIG. 13 a) or anti GRP78 antibody (FIG. 13 b) followed byChemiluminescent Substrate and confirmed the presence of GRP78 protein.

FIG. 14 is a bar graph depicting the results of FACS analysis ofendothelial cells under normoxia and hypoxia conditions using anti-GRP78antibody. The anti-GRP78 antibody was added to endothelial cellsoriginating from 10 different cords and the binding of the antibody tothe cells was determined by FACS analysis. Note the increase in percentbinding of anti-GRP78 antibody to endothelial cells under hypoxiaconditions.

FIG. 15 is a FACS histogram depicting the binding of BiotinylatedADOPep1 to endothelial cells under normoxia and hypoxia conditions. Xaxis—Intensity of binding. Binding of ADOPep^(Biot) added at 5micrograms per 100,000 cells to endothelial cells increased underhypoxia.

FIGS. 16 a-d are FACS analyses of different tumor cell lines using theanti-GRP78 antibody. Anti-GRP78 antibody was added to MCF7 breastcarcinoma (FIG. 16 a), HT-29 colon carcinoma (FIG. 16 b), SK-28 melanoma(FIG. 16 c) and K562 erytroblastoma (FIG. 16 d) and the binding to thecells was detected using FACS analysis. Note the increase in percentbinding of anti-GRP78 antibody to MCF7, HT29 and SK-28 cell lines. Alsonote that anti-GRP78 antibody did not bind to the membrane of K562cells.

FIG. 17 is a histogram depicting the effect of ADOPep1 onhypoxia-induced apoptosis of endothelial cells. Endothelial cells wereexposed for 24 hours hypoxia in the presence of endothelial cell growthmedium supplemented with 5% fetal calf serum (FCS), followed byincubation under hypoxia conditions with ADOPep1 (10 ng/ml) oranti-GRP78 antibody (100 ng/ml). Cells (100,000/tube) were incubatedwith Annexin-V FITC/PI for endothelial cell apoptosis determination.Percent apoptosis of endothelial cells stained with Annexin-V/PIdemonstrate that hypoxia conditions induce 60% apoptosis whereasaddition of ADOPep1 reduced hypoxia-induced apoptosis to 25%. Similarly,anti-GRP78 antibody induced a reduction in apoptosis up to 32%.

FIGS. 18 a-e are dot plot FACS analyses depicting the inhibition ofapoptosis by the ADOPep1. Endothelial cells under hypoxia were stainedwith both Annexin V (shown on the X axis) and Propidium Iodide (shown onthe Y axis) apoptotic markers. Cells were incubated with complete mediumunder normoxia (FIG. 18 a), in the presence of 5% FCS and completemedium under normoxia (FIG. 18 b), in starvation medium under hypoxiafor 24 hours (FIG. 18 c), in starvation medium under hypoxia for 24hours and in the presence of ADOPep1 (FIG. 18 d) or in starvation mediumunder hypoxia for 24 hours and in the presence of anti-GRP78 antibody(FIG. 18 e). Note that incubation with peptide ADOPep1 induced aremarkable decrease in the stained cells (FIG. 18 d), demonstrating itsfeasibility to reduce hypoxia-induced apoptosis.

FIG. 19 is a graph depicting inhibition of binding (in percentages) ofanti-GRP78 antibody to endothelial cells by increasing concentrations ofADOPep1, 2, and 3 peptides. Endothelial cells were incubated for 2 hourswith increasing concentrations of ADOPeps, following which the cellswere incubated for another 1 hour with the 2 μg/ml of anti-GRP78antibody and the amount of bound antibody on EC was detected using anELISA reader. Note that while increasing concentrations of ADOPep1, 2 or3 resulted in up to 50% inhibition of binding of the anti-GRP78 antibodyto endothelial cells, the sRoY scrambled peptide (SEQ ID NO:10;RYHLIPRGWDHS) exhibited no specific inhibition effect on the binding ofanti-GRP78 to endothelial cells.

FIGS. 20 a-b depict binding of the anti-GRP78 antibody to endothelialcells following incubation of the cells with ADOPep1. Endothelial cellswere incubated for 48 hours under either normoxia conditions, or hypoxiafor 24 hours followed by normoxia for another 24 hours, in the presenceor absence of the ADOPep1 (at a concentration of 10 ng/ml) and thebinding to anti-GRP78 antibody was determined using FACS analysis. FIG.20 a—a flow cytometry analysis of endothelial cells under normoxia(green plot), hypoxia (for 24 hours) followed by normoxia (for another24 hours) in the absence (red plot) or presence (blue plot) of theADOPep1 peptide. Note that while the binding of anti-GRP78 increasedfrom 18.1% under normoxia to 40.1% under hypoxia, a more significantincrease (up to 83.8%) was observed when the cells were incubated in thepresence of the ADOPep1 under hypoxia conditions. FIG. 20 b—A histogramdepicting the percent of anti-GRP78 binding to endothelial cells undernormoxia, hypoxia (for 24 hours), normoxia with 10 ng/ml ADOPep1,hypoxia (24 hours) followed by normoxia (24 hours) or hypoxia (24 hours)followed by normoxia (24 hours) in the presence of 10 ng/ml ADOPep1.Results represent average ±standard deviations of three independentexperiments.

FIG. 21 is a bar graph depicting the mean number of GRP78-positive(GRP78+) cells per area in hind limb ischemia treated with ADOPep1 as afunction of the days post induction of ischemia. Mice were subjected tohind limb ligation (the ischemic model) and after one day the mice wereinjected with either 1 μg/mice of ADOPep1, or 50 μl of PBS as control.The number of GRP78 positive cells was determined in histologicalsections prepared from ligated hind limbs (injected with ADOPep1 or PBS)of mice sacrificed at 7, 14 or 21 days post ischemia induction or normaluntreated (non-ischemic) hind limbs. Note that while the number of GRP78positive cells decreased in PBS injected hind limbs from day 7 to day 14and further at day 21 post ligation, the number of GRP78 positive cellssignificantly increased in hind limbs injected with ADOPep1 from 7 to 14days post ligation and further decreased at 21 days post ligation.

FIG. 22 is a bar graph depicting the number of apoptotic cells per fieldin ischemic hind limb treated with ADOPep1 as a function of days afterischemia induction. Mice were subjected to hind limb ligation and PBS orADOpep1 injection as described in the description of FIG. 21,hereinabove, and the number of apoptotic cells per microscopical filedwas determined in histological sections prepared from either ligated andADopep or PBS treated hind limbs of mice sacrificed at 7, 14 or 21 dayspost ischemia induction or from normal non-ischemic hind limbs. Note thesignificant decrease (7 days post ligation) in the number of ischemiainduced apoptotic cells in ligated hind limbs injected with ADOPep1 ascompared to PBS-injected ischemic hind limbs.

FIG. 23 is a graph depicting the inhibition of binding of ADOPep1^(Biot)(2 μg/ml) to endothelial cells by specific peptides corresponding toconserved motifs from the ADOPeps. Endothelial cells were incubated for2 hours under normoxia conditions in the presence of increasingconcentrations (0.01, 0.1 or 1 ng/ml) of peptides having the amino acidsequences corresponding to Motif A (HWRRP; SEQ ID NO:7), Motif B (HWRRA;SEQ ID NO:8), Motif C (AHLLP; SEQ ID NO:6) or non-biotinylated ADOPep1(SEQ ID NO:2) and then were incubated for 1 hour with biotinylatedADOPep1 (2 μg/ml) and the binding of biotinylated ADOPep1 to endothelialcells was determined using HRP-streptavidin. Note that the peptideshaving amino acid sequence corresponding to Motif A and B exhibited asignificant inhibition of the binding of ADOPep1^(Biot) to endothelialcells.

FIGS. 24 a-d are FACS analyses depicting hypoxia induced apoptosis.Endothelial cells were incubated for 24 hours under hypoxia conditionsin the absence (FIG. 24 a) or presence of 10 ng/ml of ADOPepe1 (FIG. 24b), peptide of Motif A (FIG. 24 c) or peptide of motif C (FIG. 24 d) andthe level of apoptosis was determined using FACS analysis and the PI(shown on the Y axis)/Annexin V (shown on the X axis) markers. Note thatwhile ADOPep1 and Motif A peptides were capable of inhibiting thehypoxia induced apoptosis from 79.3% under hypoxia to 28.6% (ADOPep1) or35.8% (Motif A), the motif C peptide exhibited no effect of hypoxiainduced apoptosis (81.1%).

FIG. 25 is a bar graph depicting the inhibition of hypoxia inducedapoptosis by ADOPep1, Motif A and Motif B. Endothelial cells wereincubated for 24 hours under hypoxia conditions in the absence orpresence of 10 ng/ml of ADOPepe1, Motif A, Motif B or motif C and thelevel of apoptosis was determined using FACS analysis and quantified asthe percent of Annexin V and PI positive cells. The results representaverage ±standard deviation of 4 independent experiments. Note thatwhile ADOPep1 and Motif A and B peptides exhibited a significantinhibition of hypoxia induced apoptosis, the Motif C peptide exhibitedno effect on apoptosis.

FIGS. 26 a-b are graphs depicting induction of endothelial cellmigration under hypoxia by the ADOPEP motifs. Endothelial cells wereincubated for 5 hours under hypoxia conditions in the presence ofincreasing concentrations of ADOPep1 (red line), peptide motif A (darkblue line), peptide motif B (green line) or peptide motif C (light blueline) and the migration of endothelial cells was detected. FIG. 26a-experiment 1; FIG. 26 b—experiment 2. Note that while the ADOPep1,Motif A and B peptides induced endothelial cell migration at aconcentration of about 10 ng/ml, motif C peptide exhibited nosignificant effect on endothelial cell migration.

FIG. 27 is a graph depicting induction of tube formation using AdoPep1and Motif A (SEQ ID NO:7) but not the scrambled sROY peptide.Endothelial cells were incubated in media without supplements for 24hours, and 50,000 cells were then transferred in 500 μl medium to24-well plates precoated with 250 μl Cultrex Basement Membrane Extract(with reduced growth factors) (R&D Systems, Minneapolis, Minn., USA).AdoPep1, Motif A and scrambled sROY peptides were added at an optimalconcentration of 10 ng/ml (based on preliminary findings), and theslides were examined by light microscopy after 18 hours incubation. Theresults represent average ±SD of three experiments using 3 differentcords. The length of the network of connected cells (tube formation) wasmeasured in micrometers in 5 different areas of each well usingImage-Pro Plus Image software (Media Cybernetics, Silver Spring, Md.,USA). Note that ADOPep1 and Motif A significantly increased the lengthof the network of connected cells in endothelial cells under starvationand normoxic conditions while scrambled sROY (as a control peptide), didnot induced tube formation.

FIGS. 28 a-d are FACS histograms (FIGS. 28 a-c) and a graph (FIG. 28 d)demonstrating that AdoPep1 and Motif A peptides compete on the bindingto the same receptor on endothelial cells. Endothelial cells werecultured for 24 hours under hypoxic conditions in endothelial cellgrowth medium. Cells were removed by trypsin and incubated for 1 hour onice with increasing concentrations of AdoPep1, Motif A and Motif Cpeptides. GRP78 polyclonal antibody (2 μg/100,000) was added to thecells for 2 hours on ice. Anti-goat FITC (Jackson ImmunoResearch) wasadded for 30 minutes on ice. IgG1-FITC was used as the isotype control.The samples were analyzed with a FACScan (Beckton Dickinson). Note thatAdoPep1 peptide (FIG. 28 a) and Motif A peptide (FIG. 28 b) but notMotif C peptide (FIG. 28 c) inhibit the binding of GRP78 to endothelialcells. FIG. 28 d depicts the average percent binding of GRP78 toendothelial cells as a function of peptide amount as determined by 2independent experiments.

FIG. 29 is a graph depicting GRP78 receptor internalization response toADOPep1 binding under hypoxic conditions. Endothelial cells wereincubated under starvation conditions and hypoxia over night. Cells wereincubated with 10 ng/ml ADoPep1 for 5, 15, 30 and 60 minutes. Formembrane staining biotinylated AdoePep1 at 5 micrograms was added tointact cells for 1 hour and replaced by streptavidin FITC. Forintracellular staining, cells were fixed with 1% paraformaldehydefollowed by 15 minutes incubation with 0.1% saponin. After washing ofcells, biotinylated AdoPep was added to the cells. The samples wereanalyzed with a FACScan (Beckton Dickinson). Membrane staining is shownby the blue bars (results are expressed in percentages, as normalized tountreated cells); Mean intracellular staining is shown by the purplebars (results are expressed in mean fluorescence);

FIGS. 30 a-b are Western blot analyses depicting induction ofphosphorylation of ERK by ADoPep1 and Motif A. Endothelial cells understarvation conditions and 5 hours hypoxia were incubated for 5 (lanes 2and 6), 20 (lanes 3 and 7), 30 (lanes 4 and 8) and 60 (lanes 5 and 9)minutes with 10 ng/ml ADoPep1 (Lanes 2-5) or Motif A (lanes 6-9)peptides or remained under hypoxia without a further incubation with apeptide (lane 1). After incubation, lysates were prepared, subject toSDS-PAGE and blotting on a nitrocellulose membrane. Western Blotanalysis was performed using anti-Phospho ERK1/2 antibody. Note theinduction of ERK1/2 phosphorylation after 20 minutes incubation withADoPep1 and Motif A under hypoxia conditions. Densitometry measurementsshowed a maximal ERK phosphorylation after 20 minutes incubation withADOPep1 and from 20 to 60 minutes with Motif A (data not shown). FIG. 30a—Experiment 1; FIG. 30 b—Experiment 2. The percent adjusted volume asdetermined by a densitometric analysis software to compare net banddensities was as follows: FIG. 30 a, lane 1—4.5, lane 2—9.3, lane3—13.7, lane 4—13.3, lane 5—11.2, lane 6—10.7, lane 7—12.4, lane 8—12.3and lane 9—12.4; FIG. 30 b, lane 1—4.21, lane 2—12.4, lane 3—12.5, lane4—14.8, lane 5—10.9, lane 6—14.9, lane 7—9.8, lane 8—12.7 and lane9—7.8.

FIG. 31 is a Western blot analysis depicting the level of phosphorylatedERK (pERK) in endothelial cells activated for 20 minutes with ADOPep1and motif A and inhibited by pERK-inhibitor peptide. Endothelial cells(from cord 1) under starvation conditions and 5 hours hypoxia wereincubated with 10 ng/ml ADOPep1 (lanes 3 and 4) or motif A (lanes 1 and2) peptides in the presence (lanes 2 and 4) or absence (lanes 1 and 3)of p-ERK-inhibitor peptide (Santa Cruz). For control, the endothelialcells under starvation conditions and 5 hours hypoxia (lane 5) were alsoincubated with the p-ERK-inhibitor peptide (lane 6). Note the inhibitionof ERK phosphorylation in the presence of the p-ERK-inhibitor peptide.The percent adjusted volume as determined by a densitometric analysissoftware to compare net band densities was as follows: lane 1—19, lane2—14, lane 3-14, lane 4—8.8, lane 5—11, lane 6—2.1.

FIGS. 32 a-b are Western blot analyses depicting the effect of ADOPepson the level of phosphorylated ERK. Endothelial cells (EC) from cord 1(FIG. 32 a) or cord 2 (FIG. 32 b) under hypoxia (5 hours) were incubatedfor 20 or 30 minutes with 10 ng/ml of AdoPep1, AdoPep2 and AdoPep3 orremained untreated (i.e., under hypoxia without any peptide). WesternBlot analyses of samples cell lysates were performed with anti PhosphoERK antibody on nitrocellulose membranes. FIG. 32 a: Lanes 1 and 2—ECunder hypoxia (untreated); lanes 3 and 4—EC under hypoxia incubated withADOPep3 for 20 (lane 3) or 30 (lane 4) minutes; lanes 5 and 6—EC underhypoxia incubated with ADOPep2 for 20 (lane 5) or 30 (lane 6) minutes;lanes 7 and 8—EC under hypoxia incubated with ADOPep1 for 20 (lane 7) or30 (lane 8) minutes; FIG. 32 b: Lanes 1-4—EC under hypoxia incubated for20 minutes with ADOPep1 (lane 2), ADOPep2 (lane 3), ADOPep3 (lane 4) orremained untreated (lane 1); lanes 5-8—EC under hypoxia incubated for 30minutes with ADOPep1 (lane 6), ADOPep2 (lane 7), ADOPep3 (lane 8) orremained untreated (lane 5); Densitometry measurements showed anincrease in ERK1/2 phosphorylation after incubation of endothelial cellswith ADOPep1 and 2 but not with AdoPep3. The percent adjusted volume asdetermined by a densitometric analysis software to compare net banddensities was as follows: FIG. 32 a, lane 1—11, lane 2—13, lane 3—11,lane 4—5, lane 5—11, lane 6—13, lane 7—17, lane 8—16.7; FIG. 32 b, lane1—17, lane 2—22, lane 3—19, lane 4—7, lane 5—8, lane 6—13, lane 7—14,lane 8—1.

FIGS. 33 a-f are representative FACS analyses depicting a specificinhibition of hypoxia-induced apoptosis of endothelial cells by ADOPep1.Endothelial cells in Petri dishes were incubated for 24 hours with 5%FCS in supplement-free endothelial cell growth medium under normoxia(FIGS. 33 a and d), exposed to 24 hours hypoxia (Figures c and f) orincubated under normoxia with 100 micromolar per 100,000 cells CoCl₂(FIGS. 33 b and e), in the absence (FIGS. 33 a-c) or presence (FIGS. 33d-f) of 10 ng/ml Adopep1 peptide (for 24 hours). Cells were trypsinizedand immediately re-suspended in PBS with 5% FCS and 0.1% Na-azide.Samples containing 100,000 cells were tested for apoptosis using theAnnexin-FITC (X axis) and propidium iodide (Y axis) kit (BenderMedsystems, Vienna, Austria), according to manufacturer's instructions.Results were analyzed by FACScan (Beckton Dickinson). Note that whileCoCl₂ and hypoxia conditions increased the fraction of apoptotic cells(indicated by the increase of PI and Annexin-FITC positive cells inFIGS. 33 b and c as compared to FIG. 33 a), when the cells wereincubated with ADOPep1 peptide, a significant fraction ofhypoxia-induced apoptosis was inhibited (compare PI and Annexin-FITCpositive cells in FIG. 33 f to those in FIG. 33 c), however, no effecton apoptosis induced by CoCl₂ was observed (compare PI and Annexin-FITCpositive cells in FIG. 33 e to those in FIG. 33 b).

FIG. 34 is a histogram depicting the FACS results of the experimentsdescribed in FIGS. 33 a-f. Data represents average ±SD of 2 experiments.Note that 24 hours of hypoxia or CoCl₂ treatment increased the level ofapoptosis from about 27% (under normoxia) to about 70% or 67%,respectively. Also note that ADOPep1 treatment significantly inhibitedthe hypoxia induced apoptosis by approximately 40 percent but not theCoCl₂ induced apoptosis.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Some embodiments of the invention relate to peptides which can bind toendothelial cells via the GRP78 receptor and induce angiogenesis and touses thereof for treating pathologies characterized by insufficientangiogenesis such as ischemic diseases.

The principles and operation of the peptides, compositions and methodsaccording to the invention may be better understood with reference tothe drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

While reducing the invention to practice, the present inventors haveuncovered that short amino acid sequences comprising theHistidine-Tryptophan-Arginine-Arginine (HWRR) amino acid motif (derivedfrom the ADAM15 protein) are capable of binding to the GRP78 receptor onendothelial cell, induce endothelial cells proliferation and migrationand inhibit hypoxia-induced apoptosis and thus can be used to induceangiogenesis and treat ischemic diseases.

As described in the Examples section which follows, the peptides of theinvention (e.g., ADOPeps-1, 2 and 3 as set forth by SEQ ID NOs: 2, 3 and4, respectively, or the peptides set forth by SEQ ID NOs:7 and 8) bindto endothelial cells in vitro (FIGS. 3 and 4) and induce endothelialcell proliferation (FIG. 5) and migration (FIGS. 6 and 26 a-b) underhypoxia. In addition, in vivo experiments utilizing the mouse hind limbischemia model have shown that treatment of the ischemic mice with thepeptides of the invention (e.g., ADOPep1) significantly increases bloodperfusion (FIG. 7) and blood vessel density (FIGS. 8, 9 a-b). Moreover,immuno precipitation (IP) and Western blot analyses revealed that thereceptor of ADOPeps on endothelial cells is the glucose-regulatedprotein (GRP78; SEQ ID NO:9) (FIGS. 10 a-b, 11, 12) which is expressedon various tumor cells (FIGS. 16 a-d) and that the peptides of theinvention (e.g., ADOPep1) bind to GRP78 on endothelial cells underhypoxia (FIGS. 13 a-b and 19). In addition, while hypoxia conditionsincrease the presentation of the GRP78 receptor on endothelial cells toabout 40% (FIG. 15), pre-incubation of endothelial cells with theADOPep1 further increases GRP78 presentation on endothelial cells toabout 84% (FIGS. 20 a-b, 21). Furthermore, the peptides of the invention(e.g., ADOPep1, or shorter peptides such as SEQ ID NOs: 7 and 8)prevented hypoxia-induced apoptosis of endothelial cells both in vitro(FIGS. 17, 18, 24 a-d and 25) and in vivo (FIG. 22) but not CobaltChloride-induced apoptosis (FIGS. 33 a-f and 34). In addition,incubation of endothelial cells with the peptides of the invention(e.g., SEQ ID NO:2 or 7) result in tube formation (FIG. 27) and asignificant increase in ERK1/2 phosphorylation (FIGS. 30 a-b, 31).Altogether, these results demonstrate the ability of the peptides of theinvention to induce angiogenesis in a tissue under hypoxia, and suggesttheir use for treating ischemic diseases.

Thus, according to one aspect of the invention, there is provided anisolated peptide comprising the amino acid sequence HWRR (SEQ ID NO:5),the peptide consists of 4 or 5 amino acids.

For example, the peptide according to this aspect of the invention canbe any of the peptides listed in Table 1, hereinbelow:

TABLE 1 Exemplary isolated peptides  of the invention Amino Acid SEQ IDSequence  NO: HWRRP  7 HWRRA  8 HWRRR 61 HWRRN 62 HWRRD 63 HWRRC 64HWRRQ 65 HWRRE 66 HWRRG 67 HWRRH 68 HWRRI 69 HWRRL 70 HWRRK 71 HWRRM 72HWRRF 73 HWRRS 74 HWRRT 75 HWRRW 76 HWRRY 77 HWRRV 78 AHWRR 79 RHWRR 80NHWRR 81 DHWRR 82 CHWRR 83 QHWRR 84 EHWRR 85 GHWRR 86 HHWRR 87 IHWRR 88LHWRR 89 KHWRR 90 MHWRR 91 FHWRR 92 PHWRR 93 SHWRR 94 THWRR 95 WHWRR 96YHWRR 97 VHWRR 98

According to an embodiment of the invention, the peptide of theinvention can be HWRRP (SEQ ID NO:7) or HWRRA (SEQ ID NO:8).

It will be appreciated that the invention also contemplates longerpeptides which comprise the HWRR amino acid sequence.

Thus, according to another aspect of the invention, there is provided anisolated peptide comprising an amino acid sequence HWRR as set forth bySEQ ID NO:5, with the proviso that the peptide is not SEQ ID NO:11(YPHIDSLGHWRR).

According to an embodiment of the invention, the peptide of this aspectof the invention comprises at least 4 amino acids (e.g., 4), at least 5amino acids (e.g., 5), at least 6 (e.g., 6) amino acids, at least 7(e.g., 7) amino acids, at least 8 (e.g., 8) amino acids, at least 9(e.g., 9) amino acids, at least 10 (e.g., 10) amino acids, at least 11(e.g., 11) amino acids, at least 12 (e.g., 12) amino acids, at least 13(e.g., 13) amino acids, at least 14 (e.g., 14) amino acids, at least 15(e.g., 15) amino acids, at least 16 (e.g., 16) amino acids, at least 17(e.g., 17) amino acids, at least 18 (e.g., 18) amino acids, at least 19(e.g., 19) amino acids, at least 20 (e.g., 20) amino acids, at least 21(e.g., 21) amino acids, at least 22 (e.g., 22) amino acids, at least 23(e.g., 23) amino acids, at least 24 (e.g., 24) amino acids, at least 25(e.g., 25) amino acids, at least 26 (e.g., 26) amino acids, at least 27(e.g., 27) amino acids, at least 28 (e.g., 28) amino acids, at least 29(e.g., 29) amino acids, at least 30 (e.g., 30) amino acids, at least 31(e.g., 31) amino acids, at least 32 (e.g., 32) amino acids, at least 33(e.g., 33) amino acids, at least 34 (e.g., 34) amino acids, at least 35(e.g., 35) amino acids, at least 36 (e.g., 36) amino acids, at least 37(e.g., 37) amino acids, at least 38 (e.g., 38) amino acids, at least 39(e.g., 39) amino acids, at least 40 (e.g., 40) amino acids, at least 41(e.g., 41) amino acids, at least 42 (e.g., 42) amino acids, at least 43(e.g., 43) amino acids, at least 44 (e.g., 44) amino acids, at least 45(e.g., 45) amino acids, at least 46 (e.g., 46) amino acids, at least 47(e.g., 47) amino acids, at least 48 (e.g., 48) amino acids, at least 49(e.g., 49) amino acids and no more than 50 (e.g., 50).

According to an embodiment of the invention, the peptide of this aspectof the invention comprises 50 or less amino acids, 49 or less aminoacids, 48 or less amino acids, 47 or less amino acids, 46 or less aminoacids, 45 or less amino acids, 44 or less amino acids, 43 or less aminoacids, 42 or less amino acids, 41 or less amino acids, 40 or less aminoacids, 39 or less amino acids, 38 or less amino acids, 37 or less aminoacids, 36 or less amino acids, 35 or less amino acids, 34 or less aminoacids, 33 or less amino acids, 32 or less amino acids, 31 or less aminoacids, 30 or less amino acids, 29 or less amino acids, 28 or less aminoacids, 27 or less amino acids, 26 or less amino acids, 25 or less aminoacids, 24 or less amino acids, 23 or less amino acids, 22 or less aminoacids, 21 or less amino acids, 20 or less amino acids, 19 or less aminoacids, 18 or less amino acids, 17 or less amino acids, 16 or less aminoacids, 15 or less amino acids, 14 or less amino acids, 13 or less aminoacids, 12 or less amino acids, 11 or less amino acids, 10 or less aminoacids, 9 or less amino acids, 8 or less amino acids, 7 or less aminoacids, 6 or less amino acids, 5 amino acids or 4 amino acids.

It will be appreciated that the length (“x”) of the peptide of theinvention can be any integer with a value which is at least “n” and nomore than “y”. Thus, n≦x≦y, wherein n<y and whereas “n” is an integerhaving a value between 4 to 49 and “y” is an integer having a valuebetween 5 and 50.

According to an embodiment of the invention, the peptide of this aspectof the invention comprises at least 4 and no more than 50 amino acids,at least 4 and no more than 7 amino acids, at least 5 and no more than 8amino acids, at least 6 and no more than 9 amino acids, at least 7 andno more than 10 amino acids, at least 8 and no more than 11 amino acids,at least 9 and no more than 12 amino acids, at least 7 and no more than13 amino acids, at least 12 and no more than 17 amino acids, at least 12and no more than 20 amino acids, at least 12 and no more than 30 aminoacids, at least 10 and no more than 20 amino acids, at least 8 and nomore than 30 amino acids, at least 12 and no more than 40 amino acids.

According to an embodiment of the invention, the peptide of this aspectof the invention comprises the amino acid sequence as set forth in SEQID NO:2.

According to an embodiment of the invention, the peptide of this aspectof the invention comprises the amino acid sequence as set forth in SEQID NO:3.

According to an embodiment of the invention, the peptide of this aspectof the invention comprises the amino acid sequence as set forth in SEQID NO:4.

The term “peptide” as used herein encompasses native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body or more capable of penetrating intocells. Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.

Further details in this respect are provided hereinunder. Peptide bonds(—CO—NH—) within the peptide may be substituted, for example, byN-methylated bonds (—N(CH₃)—CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—),ketomethylen bonds (—CO—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R isany alkyl, e.g., methyl, carba bonds (—CH2-NH—), hydroxyethylene bonds(—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds(—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturallypresented on the carbon atom. These modifications can occur at any ofthe bonds along the peptide chain and even at several (2-3) at the sametime.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid, such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the peptides of the invention may also includeone or more modified amino acids or one or more non-amino acid monomers(e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other less common amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables 2 and 3 below list naturally occurring amino acids (Table 2) andnon-conventional or modified amino acids (e.g., synthetic, Table 3)which can be used with the invention.

TABLE 2 Three-Letter One-letter Amino Acid Abbreviation Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid Xaa X asabove

TABLE 3 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg Penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg Penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine Mval NnbhmL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl Nmbcethylamino)cyclopropane

The peptides of the invention can be utilized in a linear form, althoughit will be appreciated that in cases where cyclization does not severelyinterfere with peptide characteristics, cyclic forms of the peptide canalso be utilized. Cyclic peptides can either be synthesized in a cyclicform or configured so as to assume a cyclic form under desiredconditions (e.g., physiological conditions).

It will be appreciated that since one of the main obstacles in usingshort peptide fragments in therapy is their proteolytic degradation bystereospecific cellular proteases, the peptides of the invention can besynthesized from D-isomers of natural amino acids [i.e., inverso peptideanalogues, Tjernberg (1997) J. Biol. Chem. 272:12601-5, Gazit (2002)Curr. Med. Chem. 9:1667-1675].

Additionally, the peptides of the invention include retro, inverso, andretro-inverso analogues thereof. It will be appreciated that complete orextended partial retro-inverso analogues of hormones have generally beenfound to retain or enhance biological activity. Retro-inversion has alsofound application in the area of rational design of enzyme inhibitors(see U.S. Pat. No. 6,261,569).

As used herein a “retro peptide” refers to peptides that are made up ofL-amino acid residues which are assembled in opposite direction to thenative peptide sequence.

Retro-inverso modification of naturally occurring polypeptides involvesthe synthetic assembly of amino acids with α-carbon stereochemistryopposite to that of the corresponding L-amino acids, i.e., D- orD-allo-amino acids in inverse order to the native peptide sequence. Arerto inverso analogue, thus, has reversed termini and reverseddirection of peptide bonds, while essentially maintaining the topologyof the side chains as in the native peptide sequence.

It will be appreciated that incorporation of any of the above-mentionedamino acid modifications including conserved changes in amino acidresidues of the peptides of the invention can be effected, as long asthe angiogenic function (e.g., endothelial cell proliferation,migration, vascular sprouting, vascularization) of the peptides of theinvention is retained. To test this, any of the angiogenesis assaysdescribed hereinbelow and in the Examples section which follows can beeffected.

The peptides of the invention may be synthesized by any techniques thatare known to those skilled in the art of peptide synthesis. For solidphase peptide synthesis, a summary of the many techniques may be foundin: Stewart, J. M. and Young, J. D. (1963), “Solid Phase PeptideSynthesis,” W. H. Freeman Co. (San Francisco); and Meienhofer, J (1973).“Hormonal Proteins and Peptides,” vol. 2, p. 46, Academic Press (NewYork). For a review of classical solution synthesis, see Schroder, G.and Lupke, K. (1965). The Peptides, vol. 1, Academic Press (New York).

In general, peptide synthesis methods comprise the sequential additionof one or more amino acids or suitably protected amino acids to agrowing peptide chain. Normally, either the amino or the carboxyl groupof the first amino acid is protected by a suitable protecting group. Theprotected or derivatized amino acid can then either be attached to aninert solid support or utilized in solution by adding the next aminoacid in the sequence having the complimentary (amino or carboxyl) groupsuitably protected, under conditions suitable for forming the amidelinkage. The protecting group is then removed from this newly addedamino acid residue and the next amino acid (suitably protected) is thenadded, and so forth; traditionally this process is accompanied by washsteps as well. After all of the desired amino acids have been linked inthe proper sequence, any remaining protecting groups (and any solidsupport) are removed sequentially or concurrently, to afford the finalpeptide compound. By simple modification of this general procedure, itis possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide, and so forth.

Further description of peptide synthesis is disclosed in U.S. Pat. No.6,472,505. One method of preparing the peptide compounds of theinvention involves solid-phase peptide synthesis, utilizing a solidsupport. Large-scale peptide synthesis is described by AnderssonBiopolymers 2000, 55(3), 227-50.

Recombinant techniques can be used when large amounts of the peptidesare required (can be used when long peptides are required). Suchrecombinant techniques are described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 andBrogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.

Briefly, a nucleic acid construct comprising a polynucleotide sequenceencoding the peptide of the invention and a promoter for directing theexpression of the polynucleotide in a host cell (e.g., a prokaryotichost cell such as E. Coli, or a eukaryotic host cell such as plant,yeast or mammalian cells) can be used along with the suitable host cellsunder conditions suitable for production of the recombinant peptide.

The peptides of the invention can be retrieved in “substantially pure”form. As used herein, “substantially pure” refers to a purity thatallows for the effective use of the peptide in the diverse applications,described herein (e.g., for therapeutic or diagnostic purposes).

Thus, the invention provides a composition-of-matter which comprises atleast one of the peptides of the invention. For example, thecomposition-of-matter of the invention may comprises one, two, three ormore of the peptides of the invention.

According to an embodiment of the invention, the peptide of theinvention is capable of binding the glucose-regulated protein (GRP78) asset forth by SEQ ID NO:9 on endothelial cells of the tissue.

According to an embodiment of the invention, the binding of peptide ofthe invention to GRP78 results in induction of angiogenesis (i.e.,inducing vascularization) in a tissue of a subject even under hypoxicconditions.

It will be appreciated that the pro-angiogenic peptides of the inventioncan be used to induce angiogenesis in a subject.

Thus, according to an additional aspect of the invention there isprovided a method of inducing angiogenesis in a subject in need thereof.The method is effected by administering to the subject a therapeuticallyeffective amount of at least one peptide of the peptides of theinvention, to thereby induce angiogenesis in the subject in needthereof.

As used herein the term “subject” refers to a mammal, such as a canine,a feline, a bovine, a porcine, an equine or a human subject.

According to an embodiment of the invention, the subject is human.

As used herein the phrase “a subject in need thereof” refers to asubject who is diagnosed with, predisposed to or suffers from anangiogenesis-dependent pathology.

As used herein the phrase “angiogenesis-dependent pathology” refers toany pathology (i.e., a condition, disease or disorder) which ischaracterized by and/or results from disregulated angiogenesis, i.e.,insufficient angiogenesis or excess of angiogenesis.

According to an embodiment of the invention, the angiogenesis-dependentpathology according to this aspect of the invention refers to apathology which is characterized by and/or results from insufficientangiogenesis. Examples include but are not limited to delayedwound-healing, delayed ulcer healing, reproduction associated disorders,arteriosclerosis, ischemic vascular disease, ischemic heart disease,myocardial ischemia, myocardial infarction, heart failure, myocardialdysfunction, myocardial remodeling, cardiomyopathies, coronary arterydisease (CAD), atherosclerotic cardiovascular disease, left maincoronary artery disease, arterial occlusive disease, peripheralischemia, peripheral vascular disease, vascular disease of the kidney,peripheral arterial disease, limb ischemia, critical leg ischemia, lowerextremity ischemia, cerebral ischemia [e.g., such as cerebral ischemiain childhood moyamoya disease (Touho H.2007, Surg Neurol. Jun 20; Epubahead of print)], cerebro vascular disease, retinopathy, retinal repair,remodeling disorder, von Hippel-Lindau syndrome, diabetes, hereditaryhemorrhagic telengiectasia, ischemic vascular disease, Buerger'sdisease, and ischemia associated with neurodegenerative disease such asParkinson's and Alzheimer's disease.

It will be appreciated that inducing angiogenesis in a tissue of asubject in need thereof can be used to treat a pathology (e.g., disease)characterized by insufficient angiogenesis (as described hereinabove) ina tissue of a subject.

Induction of angiogenesis and/or treating a pathology characterized byinsufficient angiogenesis in a subject is achieved according to thisaspect of the invention by administering at least one of the peptides ofthe invention to the subject.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

It will be appreciated that the peptide of the invention can beadministered to the subject per se or in a pharmaceutical compositionwhere it is mixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein (i.e., at leastone peptide of the peptides of the invention) with other chemicalcomponents such as physiologically suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Herein the term “active ingredient” refers to the peptide of theinvention accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Conventional approaches for drug delivery to the CNS include:neurosurgical strategies (e.g., intracerebral injection orintracerebroventricular infusion); molecular manipulation of the peptide[e.g., production of a chimeric fusion peptide that comprises thepeptide of the invention which can bind the surface of endothelial cellsin combination with an agent that is itself incapable of crossing theblood brain barrier (BBB)] in an attempt to exploit one of theendogenous transport pathways of the BBB; pharmacological strategiesdesigned to increase the lipid solubility of the peptide (e.g.,conjugation of a water-soluble peptide to lipid or cholesterolcarriers); and the transitory disruption of the integrity of the BBB byhyperosmotic disruption (resulting from the infusion of a mannitolsolution into the carotid artery or the use of a biologically activeagent such as an angiotensin peptide).

Alternately, one may administer the pharmaceutical composition in alocal rather than a systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the invention may be manufactured byprocesses well known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the inventionthus may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, such as in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the invention are conveniently delivered in the form of anaerosol spray presentation from a pressurized pack or a nebulizer withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in a dispenser may be formulated containing apowder mix of the compound and a suitable powder base such as lactose orstarch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuosinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the invention may also be formulatedin rectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the inventioninclude compositions wherein the active ingredients are contained in anamount effective to achieve the intended purpose. More specifically, atherapeutically effective amount means an amount of active ingredients(the peptide of the invention) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., a pathology associated withinsufficient angiogenesis) or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to providetissue (e.g., cardiac tissue, brain tissue, limb tissue, renal tissue)levels of the active ingredient are sufficient to induce the biologicaleffect (angiogenesis) (minimal effective concentration, MEC). The MECwill vary for each preparation, but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the invention may, if desired, be presented in a pack ordispenser device, such as an FDA approved kit, which may contain one ormore unit dosage forms containing the active ingredient. The pack may,for example, comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions foradministration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

Due to their selective binding to endothelial cells, the peptides of theinvention can be used to target agents fused thereto to endothelialcells (ECs) in vitro, ex vivo (i.e., in cells which were removed from asubject) and/or in vivo (i.e., within a living organism, subject). Suchan agent can be any molecule that targeting thereof to endothelial cellsis desirable and/or beneficial (e.g., beneficial to the subject when invivo administration is effected).

For example, such an agent can be a drug, a toxic moiety (e.g., which isdesigned to kill endothelial cells), a chemotherapeutic agent (which isdesigned to kill cancerous cells within or in the vicinity of theendothelial cells), an identifiable agent (e.g., biotin, digoxeginin,enzymatic moiety which can be used to detect endothelial cells), and aradio-isotope (which is capable of labeling and/or killing endothelialcells).

Examples of toxins which can be fused to the peptide of the inventioninclude, but are not limited to, enzymatically active toxins ofbacterial, fungal, plant, or animal origin, or fragments thereof [e.g.,diphteria toxin, exotoxin A chain of Pseudomonas aeruginosa, ricin Achain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes].

Examples of radioisotopes which can be fused to the peptide of theinvention include, but are not limited to, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹²Bi,¹⁹⁸Re, ¹⁸⁸Re, ¹⁸⁶Re, ²¹¹At, ⁶⁷Cu, and ²¹²Pb.

Examples of chemotherapeutic agents which can be fused to the peptide ofthe invention include, but are not limited to, Nitrogen Mustards [e.g.,Mechlorethamine (HN₂), Cyclophosphamide, Ifosfamide, Melphalan,Chlorambucil and Estramustine], alkylating agents, folic acidantagonists or analogs (e.g., Methotrexate, Trimetrexate),anti-metabolites of nucleic acid metabolism, antibiotics (e.g.,Dactinomycin, Daunorubicin, Doxorubicin, 4′-Deoxydoxorubicin, Bleomycin,Plicamycin, Mitomycin), pyrimidine analogs (e.g., Fluorouracil,Floxuridine, Cytarabine), 5-fluorouracil, cisplatin, purine nucleosides,analogs and related inhibitors (e.g., Azacitidine, Mercaptopurine,Thioguanine, Pentostatin, Fludarabine), amines, amino acids, triazolnucleosides, corticosteroids, Ethylenimines and Methylmelamines (e.g.,Hexamethyl-melamine, Thiotepa), Alkyl Sulfonates (e.g., Busulfan),Nitrosoureas (e.g., Carmustine, Lomustine, Semustine, Streptozocin),Triazenes (e.g., Dacarbazine, Procarbazine, Aziridine) Vinca Alkaloids[e.g., Vinblastine (VLB), Vincristine, Vindesine], Epipodophyl-Lotoxins(e.g., Etoposide, Teniposide), enzymes (e.g., L-Asparaginase), Taxanes(e.g., Docetaxel, Paclitaxel), biological response modifiers (e.g.,interferon alfa, tumor necrosis factor, tumor infiltrating lymphocytes),Platinum coordination complexes (e.g., Cisplatin, carboplatin),Anthracenedione (e.g., mitoxantrone), substituted urea (hydroxyurea),methyl hydrazine derivative (procarbazine), adrenocortical suppressant(mitotane, aminoglutethimide), costeroids, progestins (e.g.,hydroxy-progesterone caproate, medroxy progesterone). Specific examplesinclude, Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside(i.e., Ara-C), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol,Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No.4,675,187), Melphalan, and other related nitrogen mustards. Alsoincluded in this definition are hormonal agents that act to regulate orinhibit hormone action on tumors, such as estrogens (e.g.,diethylstilbestrol, ethinyl estradiol), antiestrogen (tamoxifen),androgens (testosterone propionate, fluoxymesterone), antiandrogen(flutamide), Gonadotropin-Releasing hormone analog (Leuprolide,Goserelin), and onapristone.

Fusions between the peptides of the invention and the abovedescribedagent can be generated using a variety of bifunctional protein-couplingagents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP)(e.g., essentially as described in Cumber et al. 1985, Methods ofEnzymology 112: 207-224), iminothiolane (IT), bifunctional derivativesof imidoesters (such as dimethyl adipimidate HCL), active esters (suchas disuccinimidyl suberate), aldehydes (such as glutareldehyde;essentially as described in G. T. Hermanson, 1996, “AntibodyModification and Conjugation, in Bioconjugate Techniques, AcademicPress, San Diego), bisazido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene) or carbodiimide conjugation procedure(as described in J. March, Advanced Organic Chemistry: Reaction's,Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985; B. Neiseset al. 1978, Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al.Tetrahedron Lett. 4475; E. P. Boden et al. 1986, J. Org. Chem. 50:2394or and L. J. Mathias 1979, Synthesis 561). For example, a ricin fusioncan be prepared as described in Vitetta et al., Science, 238: 1098(1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the peptide. See WO94/11026; U.S. Pat.No. 6,426,400; Laske, D. W., Youle, R. J., and Oldfield, E. H. (1997)Tumor regression with regional distribution of the targeted toxinTF-CRM107 in patients with malignant brain tumors. Nature Medicine3:1362-1368.

Additionally or alternatively, the agent of the invention can beattached to the peptide of the invention via recombinant DNA technologyby constructing an expression vector which comprises the coding sequenceof the agent of the invention (e.g., the PE38 KDEL truncated form ofpseudomonas exotoxin A) translationally fused to the coding sequence ofthe peptide of the invention (e.g., SEQ ID NO:2) and expressing theconstruct in a host cell (e.g., a prokaryotic or eukaryotic cell) forthe production of a recombinant fusion peptide comprising the aminoacids of the agent and the peptide of the invention. Alternatively, theexpression vector can be administered to the subject in need of therapyvia known gene therapy techniques (e.g., using vial vehicles).

According to an embodiment of the invention, the composition of theinvention which comprises the peptide of the invention and the agent(e.g., the toxin, chemotherapeutic, identifiable moiety orradio-isotope) fused (or attached) thereto can form part of apharmaceutical composition with a pharmaceutically acceptable carrier.

Accordingly, such a composition (which comprises the therapeutic agentattached or fused to the peptide of the invention) can be used fortreating a pathology characterized by abnormally increased angiogenesis(hyper-vascularization). For example, such compositions can be used toinhibit tumor growth by destruction of the tumor vasculature.

Non-limiting examples of pathologies characterized by abnormallyincreased angiogenesis which can be treated by the composition of theinvention include cancer, metastatic cancer, myelodysplastic features(MDF) in bone marrow of HIV patients, primary myelodysplastic syndromes(MDS), Systemic mastocytosis (SM), retinal neovascularization,neovascularization in atherosclerotic plaques, hemangiomas, arthritis,psoriasis, arthritis and other autoimmune diseases.

The cancer or cancer metastases which can be treated by the compositionof the invention include, but is not limited to, tumors of thegastrointestinal tract (colon carcinoma, rectal carcinoma, colorectalcarcinoma, small and/or large bowel carcinoma, esophageal carcinoma,stomach carcinoma, pancreatic carcinoma), gallbladder carcinoma, Biliarytract tumors, prostate cancer, renal cancer (e.g., Wilms' tumor), livercancer (e.g., hepatoblastoma, hepatocellular carcinoma), bladder cancer,embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor,testicular germ cells tumor, immature teratoma of ovary, uterine,epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placentalsite trophoblastic tumor, epithelial adult tumor, ovarian carcinoma,cervical carcinoma, small-cell and non-small cell lung carcinoma,nasopharyngeal, breast carcinoma, squamous cell carcinoma (e.g., in headand neck), neurogenic tumor, astrocytoma, ganglioblastoma,neuroblastoma, lymphomas (e.g., Hodgkin's disease, non-Hodgkin'slymphoma, B cell, Burkitt, cutaneous T cell, histiocytic, lymphoblastic,T cell, thymic), gliomas, adenocarcinoma, adrenal tumor, brainmalignancy (tumor), various other carcinomas (e.g., bronchogenic largecell, ductal, Ehrlich-Lettre ascites, epidermoid, large cell, Lewislung, medullary, mucoepidermoid, oat cell, small cell, spindle cell,spinocellular, transitional cell, undifferentiated, carcinosarcoma,choriocarcinoma, cystadenocarcinoma), ependimoblastoma, epithelioma,erythroleukemia (e.g., Friend, lymphoblast), fibrosarcoma, giant celltumor, glial tumor, glioblastoma (e.g., multiforme, astrocytoma), gliomahepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g.,B cell), hypernephroma, insulinoma, islet tumor, keratoma,leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphatic, acutelymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic Tcell leukemia, acute—megakaryoblastic, monocytic, acute myelogenous,acute myeloid, B cell, basophilic, chronic myeloid, chronic, B cell,eosinophilic, Friend, granulocytic or myelocytic, hairy cell,lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage,myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell,promyelocytic, subacute, T cell, lymphoid neoplasm), lymphosarcoma,melanoma, mammary tumor, mastocytoma, medulloblastoma, mesothelioma,metastatic tumor, monocyte tumor, multiple myeloma, myelodysplasticsyndrome, myeloma, nephroblastoma, nervous tissue glial tumor, nervoustissue neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma,osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), papilloma,transitional cell, pheochromocytoma, pituitary tumor, plasmacytoma,retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's, histiocyticcell, Jensen, osteogenic, reticulum cell), schwannoma, subcutaneoustumor, teratocarcinoma (e.g., pluripotent), teratoma, testicular tumor,thymoma and trichoepithelioma.

A growing body of evidence indicates that angiogenesis is essential tothe progression of cancer. In fact, the extent of neovascularity isstrongly correlated with metastases in primary breast carcinoma, bladdercancer, prostrate cancer, non-small cell lung cancer, cutaneousmelanomas, and uterine cervix carcinoma [Ferrara, N., Breast CancerResearch and Treatment 36: 127-137 (1995)]. Thus, assessing theangiogenic phenotype of tumors will provide a strong indication todisease outcome. Other diseases or conditions which are characterized byhypervascularization or hypovascularization include, but are not limitedto, retinal neovascularization, neovascularization in atheroscleroticplaques, hemangiomas, arthritis, and psoriasis, as well as the diseasesdescribed hereinabove. See Folkman, J. New England J. of Med.333:1757-63 (1995).

Thus, the ability of the peptides of the invention to bind specificallyto the cell-surface of endothelial cells, suggests the use thereof aspotent detectors of vascularization. This may be important for detectingthe presence of, assessing predisposition to, or monitoring progressionof angiogenesis-dependent diseases.

Thus, the invention also envisages a method of detecting a presence oran absence of endothelial cells in a biological sample.

The method is effected by incubating the biological sample with thepeptide of the invention (which is capable of binding to thecell-surface of endothelial cells, e.g., via the GRP78 receptor) anddetecting the peptide, to thereby detect the presence or the absence ofendothelial cells in the biological sample.

The biological sample utilized for detection can be a tissue sample suchas a biopsy specimen. Methods of obtaining tissue biopsies from mammalsare well known in the art (see Hypertext Transfer Protocol://World WideWeb dot healthatoz dot com/healthatoz/Atoz/default dot html).

At least one peptide of the invention is contacted with the biologicalsample under conditions suitable for complex formation (i.e., buffer,temperature, incubation time etc.) as described in the Examples sectionwhich follows.

Peptide-cell complexes within a biological sample can be detected viaany one of several methods known in the art, which methods can employbiochemical and/or optical detection schemes.

To facilitate complex detection, the peptides of the invention arehighlighted by a tag or an antibody. It will be appreciated thathighlighting can be effected prior to, concomitant with or followingcomplex formation, depending on the highlighting method. As used hereinthe term “tag” refers to a molecule, which exhibits a quantifiableactivity or characteristic. A tag can be a fluorescent moleculeincluding chemical fluorescers, such as fluorescein or polypeptidefluorescers, such as the green fluorescent protein (GFP) or relatedproteins (www dot clontech dot corn). In such case, the tag can bequantified via its fluorescence, which is generated upon the applicationof a suitable excitatory light. Alternatively, a tag can be an epitopetag, a fairly unique polypeptide sequence to which a specific antibodycan bind without substantially cross reacting with other cellularepitopes. Such epitope tags include a Myc tag, a Flag tag, a His tag, aleucine tag, an IgG tag, a streptavidin tag and the like.

It will be appreciated that the peptides of the invention may also beused as potent detectors of endothelial cells in vivo. A designedpeptide capable of binding endothelial cells, labeled non-radioactivelyor with a radio-isotope, as is well known in the art can be administeredto an individual to diagnose the onset or presence ofangiogenesis-dependent disease, discussed hereinabove. The binding ofsuch a labeled peptide after administration to endothelial cells can bedetected by in vivo imaging techniques known in the art.

It will be appreciated that the peptide of the invention can be furtherused to identify a putative angiogenic molecule (i.e., a moleculecapable of inducing angiogenesis) or anti-angiogenic (i.e., a moleculecapable of inhibiting angiogenesis). Thus, according to another aspectof the invention there is provided a method of identifying a putativeangiogenic molecule. The method is effected by: (a) providingendothelial cells having the peptide of the invention bound thereto, and(b) identifying a molecule capable of displacing the peptide from theendothelial cells, to thereby identify a putative angiogenic oranti-angiogenic molecule.

Alternatively, the method of identifying the putative angiogenic oranti-angiogenic molecule can be effected by: (a) incubating the peptideof the invention with a glucose-regulated protein (GRP78) or cellsexpressing the GRP78 under conditions suitable for formation of acomplex between the peptide and the GRP78 or the cells expressing GRP78,and (b) identifying a molecule capable of displacing the peptide fromthe complex, to thereby identify a putative angiogenic oranti-angiogenic molecule.

It will be appreciated that such a detection method can also be utilizedin an assay for uncovering potential drugs useful in inhibition orpromotion (induction) of angiogenesis. For example, the invention may beused for high throughput screening of test compounds (i.e., putativeangiogenic or anti-angiogenic molecules). Typically, the peptides of theinvention are radiolabeled, to reduce assay volume. The peptides areallowed to bind endothelial cells prior to, concomitant with orfollowing binding of the test compound. A competition assay is theneffected by monitoring displacement of the label by a test compound [Han(1996) J. Am. Chem. Soc. 118:4506-7 and Esler (1996) Chem. 271:8545-8].

Once a putative angiogenic or anti-angiogenic molecule is identified itis further evaluated using angiogenesis assays which are well known inthe art. Examples include, but are not limited to, the chickchorioallantoic membrane, rabbit cornea assay, sponge implant models,matrigel and tumor models (see also the assays described in the Examplessection which follows).

The peptides of the invention can be included in a diagnostic ortherapeutic kit. For example, the peptides can be packaged in one ormore containers with appropriate buffers and preservatives and used fordiagnosis or for directing therapeutic treatment. Thus, the peptides,for example, can be each mixed in a single container or placed inindividual containers. According to an embodiment of the invention, thecontainers include a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials, such as glass or plastic.

In addition, other additives, such as stabilizers, buffers, blockers andthe like may also be added.

The peptides of such kits can also be attached to a solid support, suchas beads, array substrate (e.g., chips) and the like and used fordiagnostic purposes.

Peptides included in kits or immobilized to substrates may be conjugatedto a detectable label, such as described hereinabove.

The kit can also include instructions for determining if the testedsubject is suffering from, or is at risk of developing, a condition,disorder, or disease associated with disregulated angiogenesis.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the invention willbecome apparent to one ordinarily skilled in the art upon examination ofthe following examples, which are not intended to be limiting.Additionally, each of the various embodiments and aspects of theinvention as delineated hereinabove and as claimed in the claims sectionbelow finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Methods

Peptide synthesis—ADOPep1 (SEQ ID NO:2), ADOPep2 (SEQ ID NO:3) andADOPep3 (SEQ ID NO:4) were synthesized by SynPep (Dublin, Calif., USA)according to the ADAM15 amino acid sequence (GenBank Accession No.Q13444). HPLC purity was greater than 97%. The peptides were dissolvedin water in a concentration of 1 mg/ml.

Binding of ADOPep to endothelial cells—Human umbilical endothelial cells(HUVEC) were harvested by trypsin and 100,000 cells per sample weresuspended in PBS+5% FCS+0.1% Na azide. Endothelial cells were eitherexposed for 5 hours to hypoxia or remained under normoxia conditions.Cells were stained for 2 hours on ice in dark with 0.05, 0.5, 5μg/100,000 cells biotinylated ADOPeps. The cells were then washed twicewith PBS, following which the cells were stained for 30 minutes withFITC-labeled Streptavidin (Jackson ImmunoResearch Laboratories, PA,USA). After washing, samples were analyzed using a fluorescenceactivated cell sorter (FACScan Beckton Dickinson, CA, USA).

Proliferation of endothelial cells incubated with ADOPeps—HUVEC frompassage 3 were used for proliferation experiments. Endothelial cells(15,000 cells/well) were seeded on 24-wells in the presence ofendothelial cell growth medium (Promocell, Heidelberg, Germany) withsupplements and incubated for 24 hours. After then, cells were exposedto 24 hours starvation in a medium free of supplements. ADOPeps wereadded at concentrations of 1, 10 and 100 ng/ml for 24 hours underhypoxic conditions. 2 μCi/well of Thymidine (SIGMA, Rehovot Israel) wereadded or over night incubation followed by 3 washes with PBS beforeharvesting at 37° C. with 300 μl/well of 0.5 M NaOH. Cell lysates weretransferred to scintillation vials with 2 ml scintillation liquid UltimaGold (Packard Bioscience, Meriden, USA) and counted in a counter.Results were obtained as counts per minute (cpm).

Migration of endothelial cells induced by ADOPeps—Endothelial cellmigration was evaluated by the Chemicon QCM 96-well Migration Assay(Chemicon International, CA, USA) according to manufacturer'sinstructions. This kit utilizes a membrane with an 8 μm pore size.Migratory cells on the bottom of the insert membrane are dissociatedfrom the membrane when incubated with cell detachment buffer provided bythe kit. These cells are subsequently lysed and detected by a molecularprobe CyQuant GR dye, a green fluorescent dye which exhibits fluorescentenhancement when bound to cellular nucleic acid. For the migrationassay, HUVEC from passage 3 were incubated in endothelial cell (EC)growth medium free of supplements. After trypsinization, 25,000 EC wereincubated in the migration chamber. ADOPeps were added at concentrationsof 1, 10 and 100 ng/ml in the feeder tray for chemoattractant migrationassay. Time of incubation was 5 hours for endothelial cells underhypoxic conditions. Results were determined in a Fluorescent ELISAreader at 480/520 nm.

Mouse hind-limb ischemia model for evaluation of the in vivo angiogenicactivity of the ADOPeps—A mouse hind limb ischemia model was used.Ischemia was created in the C57B1 strain mouse by ligation and excisionof the femoral artery on the left hind limb. The right hind limb servedas a control. A day after the operation ADOPep1 and 3 were injectedintramuscular at a site close to the operation. Each mouse was treatedone day after operation, with either peptide in a total amount of 0.1 or1 μg per mouse or PBS (50 μl) which was injected as a control. The bloodperfusion was measured using a Laser Doppler Blood perfusion analyzer(Perimed, Sweden) one day post operation and 7, 14, 21 days postoperation. The average perfusion of each limb was computed and percentperfusion ratio was expressed as the ischemic (left)/control (right)percent perfusion ratio.

Histological examination—Limbs from mice treated with ADOPep1, 3 orcontrol mice injected with PBS were sacrificed at 7, 14 and 21 days postoperation. Whole ischemic and non-ischemic legs were immediately fixedfor 48 hours in 4% paraformaldehyde and then embedded in paraffin.Three-micrometer thick sections were prepared and cut with the musclefibers oriented transversely. Identification of endothelial cells wasperformed by immunostaining for von Willebrand Factor VIII relatedantigen using a primary antibody of polyclonal anti-factor VIII at a1/200 dilution (DakoCytomation, Denmark) and secondary antibody theEnvision+System-HRP (DakoCytomation, Denmark). Vessel densities wereexpressed as capillaries per millimeter squared. To obtain the averagevessel number per cross-section area, a minimum of ten individual fieldswere sampled, and Image Pro-Plus (MediaCybernetics, Silver spring, MD,USA) was used to measure the counted area. The number of Factor VIIIpositive vessels was counted.

Immune Precipitation (IP) Experiments of Endothelial Cells withBiotinylated ADOPep

Preparation of HUVEC lysates—HUVEC were seeded in 90 mm Petri dishesplates for 24 hours with complete Endothelial Cell Growth Medium(PromoCell. Heildelberg, Germany). Cells were washed twice with PBS andbuffer lysate (50 mM Tris Cl (pH 8, 150 mM Na Cl, 0.02% Na azide, 0.1%SDS, 100 μg/ml PMSF, 1 μg/ml protease inhibitors, 1% NP-40) was addedfor 20 minutes on ice. After incubation, cells were scraped with arubber policeman and transferred to a chilled microfuge tube. Aftercentrifugation of lysate at 12,000 g for 2 minutes at 4° C., supernatantwas transferred to a fresh microfuge tube and stored at −70° C.

Immunoprecipitation (IP)—Aliquots samples of lysates (500 μl) wereincubated with 10 μg of biotinylated ADOPep1 for one hour at 4° C. bygently mixing. Streptavidin Sepharose beads (Amersham Biosciences,Uppsla, Sweeden) (50 μl) were added to the sample for a secondincubation one hour at 4° C. by gently mixing. After then, sample wascentrifuge for 20 seconds at 12,000 g and the pellet was saved andfurther washed 3 times. Finally the pellet was suspended in 100 μl ofsample buffer for Western blot analysis. For ELISA, protein was elutedby 5 minutes incubation of the sample at room temperature in 1 ml ofTris-Glycin 0.2 M (pH 2.2), followed by titration with 1 M Tris (pH9.1).

Polyacrylamide Gel Electrophoresis (PAGE) analysis—The protein bandcontaining GRP78 which was immunoprecipitated with the biotinylatedADOPep1 (GRP78 immune-precipitated protein) (30 μl) was applied tominigel lane and run at standard conditions (60 mA for 2 minigels, 1.4hours) in a 10% Tris Acrylamide gel. Gel was stained with Coomassieblue. Band was cut and sent to mass-spectroscopy for proteinidentification.

Western Blot analysis—Following PAGE analysis, proteins transfer wasperformed for 2 hours in wet conditions at 40 V in a nitrocellulosemembrane, following which the gel was stained with Coomassie blue. Thenitrocellulose membrane was blocked for 2 hours at room temperature byincubation with PBS containing 0.5% Tween-20 and 5% non-fat milk.Incubation of the membrane with biotinylated ADOPep1 (5 μg/ml inPBS-Tween) was performed over night at 4° C. by gently shaking. Afterthen, membrane was washed 3 times (15 minutes each) in PBS-Tween.Incubation with the secondary antibody Peroxidase-conjugatedStreptavidin (1 μg/ml) (JacksonImmunoResearch, PA, CA USA) was performedfor 45 minutes at room temperature followed by 3 washes (15 minuteseach) in PBS-Tween. For GRP78 immunostaining, the membrane was blockedas described hereinabove and further incubated for 24 hours at 4° C.with anti-GRP78 antibody (Santa Cruz Biotechnologies, CA, USA) at aconcentration of 2 micrograms per ml, followed by 3 washes withPBS-Tween. Incubation with a secondary anti-goat FITC (JacksonImmunoResearch Laboratories, PA, USA) at a dilution of 1:5000 wasperformed for 45 minutes at room temperature, followed by 3 washes inPBS-Tween. ECL was performed using SuperSignal West PicoChemiluminescent Substrate (Pierce, Ill., USA).

FACS analysis of anti GRP78 binding to Endothelial Cells—HUVEC wereharvested by trypsin and 100,000 cells per sample were suspended inPBS+5% FCS+0.1% Na azide. Goat polyclonal anti GRP78 (Santa CruzBiotechnologies, CA, USA) at a concentration of 1 μg/100,000 cells wasadded for 40 minutes on ice. Cells were washed and stained withanti-goat FITC (Jackson ImmunoResearch Laboratories, PA, USA). Sampleswere analyzed using a fluorescence activated cell sorter (FACScanBeckton Dickinson, CA, USA).

FACS analysis of anti GRP78 binding to different tumor cells—MCF7 breastcarcinoma, SK28 Melanoma, HT 29 colon carcinoma and K562 erythroleukemiawere harvested by trypsin and 300,000 cells per sample were suspended inPBS+5% FCS+0.1% Na azide. Goat polyclonal anti GRP-78 (Santa CruzBiotechnologies, CA, USA) at a concentration of 1 μg/100,000 cells wasadded for 40 minutes on ice. Cells were washed and stained withanti-goat FITC (Jackson ImmunoResearch Laboratories, PA, USA). Sampleswere analyzed using a fluorescence activated cell sorter (FACScanBeckton Dickinson, CA, USA).

Apoptosis—EC were incubated for 24 hours with 5% FCS in EC growth mediain Petri dishes plates followed by 24 hours incubation under hypoxiaconditions with ADOPep1 (10 ng/ml), anti GRP78 antibody (Santa CruzBiotechnology, CA, USA) (1 microgram/ml) or recombinant VEGF (10 ng/ml).The Annexin V FITC/PI detects the phosphatidylserin on the apoptoticcells using flow cytometry. Human Annexin V-FITC kit (Bender Medsystems,Vienna, Austria) was used for the measurement of EC apoptosis percentagefollowing manufacturer's instructions. Samples were analyzed using afluorescence activated cell sorter (FACScan Beckton Dickinson, CA, USA).

Competitive binding of ADOPep1, ADOPep2 and ADOPep3 peptides toGRP78—Endothelial cells (20,000 per well) were seeded in 96 well platesfor 24 hours in the presence of the complete medium. Plates were washedwith PBS over night and rehydrated with PBS, 0.1% Na Azide and 5% FCS.ADOPep1, ADOPep2 and ADOPep3 were added to washed plates for 2 hours atroom temperature at concentrations of 0.01, 0.1 and 1 microgram per ml.Anti-GRP78 antibody (goat polyclona IgG, Santa Cruz Biotechnology, CA,USA) was added to the plates for 1 hour at room temperature at aconcentration of 2 micrograms/ml per well. After washing, boundanti-GRP78 antibody was detected by incubation with anti-goat IgGPeroxidase conjugated (Jackson Immuneresearch Laboratories, PA, USA).After 5 washes with PBS-0.1% Tween 20, 100 μl/well of TMB+Substrate-Chromogen (DAKOCytomation, CA, USA) was added for a maximum of30 minutes. Reaction was stopped with 1 N HCl. Color developed wasdetermined by an ELISA reader at 450 nm.

Example 1 Specific Peptides Derived from the Metalloprotease Domain ofADAM15 Bind to Endothelial Cell and Induce Proliferation and MigrationThereof

Experimental Results

ADOPep1 Sequence—Several peptides were synthesized from themetalloprotease domain and preliminary experiments were conducted inorder to select a peptide with the best binding ability to endothelialcells. One of these, a peptide termed ADOPep1, has the amino acidsequence set forth by SEQ ID NO:2, HWRRAHLLPRLP. Its location in themetalloprotease domain of ADAM15 molecule (SEQ ID NO:1) is presented inFIG. 2 b (underlined text corresponding to amino acids 286-297 of SEQ IDNO:1).

Binding of ADOPep1 to endothelial cells (EC) by FACS analysis—Increasingconcentrations of biotinylated ADOPep1 were added to EC under normoxiaconditions and the binding of ADOPep1 to EC was detected usingFITC-labeled Streptavidin and FACS analysis. As can be seen in FIG. 3 anincrease in percent binding of ADOPep1 to EC reached about 62% in a dosedependent manner and was maximal at 5 micrograms per ml. When EC underhypoxia conditions (for 5 hours) were incubated with biotinylatedADOPep1, the binding of the ADOPep1 at 5 μg/ml reached about 85% (FIG.4). These results demonstrate an increase in binding of ADOPep1 toendothelial cells under hypoxia.

ADOPeps induce proliferation of EC under hypoxia—To further test theability of ADOPeps to induce EC proliferation, increasing concentrationsof ADOPeps 1, 2 and 3 were incubated with EC and the proliferation ofcells under hypoxia and 24 hours starvation was determined. As shown inFIG. 5, ADOPep1 and ADOPep2 were capable of inducing proliferation of ECat a concentration of 10 ng/ml.

Novel peptide ADOPep1 induces migration of EC under hypoxia—The abilityof ADOPeps to induce migration of endothelial cells under hypoxia wastested. EC were incubated in the migration chamber and ADOPeps wereadded at 1, 10 and 100 ng/ml in the feeder tray for 5 hours underhypoxia conditions. The migration of EC was determined in a FluorescentELISA reader and is expressed as Relative Fluorescent Units (RFU). Asshown in FIG. 6, the most significant increase in EC migration wasobserved in the presence of ADOPep1 at a concentration of 10 ng/ml.

Altogether, these findings demonstrate that the ADOPeps of the inventionare capable of binding to endothelial cell, mainly under hypoxia andinducing proliferation and migration of endothelial cells under hypoxia.

Example 2 ADOPep1 Induces Angiogenesis and Increased Perfusion ofIschemic Tissues In Vivo

Experimental Results

ADOPep1 induces a significant increase in perfusion in mice with hindlimb ischemia—A mouse ischemic hind limb model was used for evaluationof the in vivo potential of angiogenesis induced by ADOPeps. Ischemiawas created in the mouse left hind limb by ligation and excision of thefemoral artery. The right hind limb served as control. A day after theoperation each of the peptides was injected into one site close to theligation. Each mouse was treated with each of the peptides in a totalamount of 0.1 or 1 μg per mouse. The blood perfusion was measured usinga Laser Doppler Imager (PeriMed, Sweden) at days 7, 14 and 21 afteroperation. As can be seen in FIG. 7 the average perfusion of each limbwas computed and expressed as the ischemic (left)/control (right) bloodperfusion ratio. A statistical analysis demonstrates a significantincrease in the blood perfusion ratio in mice injected with 0.1microgram ADOPep1 at day 21 after operation in comparison to miceinjected with PBS demonstrating complete recovery of the blood perfusionin the hind limb.

Histological assessment of angiogenesis in the ischemic hind limbtreated with ADOPep1—After hind limb ligation and administration of 0.1μg/per mouse of ADOPep1, the mice were sacrificed at days 7, 14 and 21and the whole legs were embedded in paraffin. FIG. 8 shows the averageof vessel number per cross section area of ten individual fields persample expressed as mean±SE in legs of mice treated with ADOPep1 incomparison to PBS injected mice. ADOPep1 treatment resulted in asignificant increase (p<0.05) in the number of blood vessels incomparison to PBS treated mice. Representative illustrations (FIGS. 9a-b) show higher von Willebrand Factor Positive stained small vessels inthe ADOPep1 treated group than in the control.

Altogether, these findings demonstrate that the ADOPeps of the inventionare capable of inducing angiogenesis following ischemia and thus can beused to treat ischemic diseases.

Example 3 Identification of the ADOPeps Receptor on Endothelial Cells

Experimental Results

Identification of the ADOPep receptor on endothelial cells—Immuneprecipitation (IP) of endothelial cells lysate with biotinylated ADOPep1was analyzed by PAGE. As can be seen in FIGS. 10 a-b, followingimmunoprecipitation with ADOPep1 a major single protein band is presentat 78 kDa. In order to confirm that this band is the ADOPep1 peptidebinding receptor, the separated proteins were transferred to anitrocellulose membrane which was further stained with biotinylatedADOPep1 followed by Chemiluminescent Substrate. As can be seen in FIG.11, indeed the same band was stained by the labeled peptide in twodifferent experiments (both lanes in the PAGE shown in FIG. 11). Theprotein receptor band was cut from the gel and analyzed bymass-spectrometry.

The results of mass-spectrometry are presented in Table 4, hereinbelow.The receptor was identified as the glucose-regulated protein [Homosapiens] GRP78 protein (GenBank Accession No. CAB71335; Gi: 6900104)with 22 peptides digested from the isolated band.

TABLE 4 IP with ADOPep1 in Normoxia IP with ADOPep 1  SEQ in Normoxia IDScore Mass Sequence NO:  1 88 1528.7 AKFEELNM(+16)DLFR 12  2 96 1588.8KSDIDEIVLVGGSTR 13  3 96 1191.6 VYEGERPLTK 14  4 96 88 1217.6DAGTIAGLNVMR 15  5 92 92 1228.6 VEIIANDQGNR 16  6 95 86 1233.6DAGTIAGLNVM(+16)R 17  7 87 1256.6 M(+16)KETAEAYLGK 18  8 95 1888VTHAVVTVPAYFNDAQR 19  9 79 1934 DNHLLGTFDLTGIPPAPR 20 10 97 1313.6FEELNMDLFR 21 11 99 1316.6 NELESYAYSLK 22 12 92 83 1329.6FEELNM(+16)DLFR 23 13 95 1397.8 ELEEIVQPIISK 24 14 99 1430.7TWNDPSVQQDIK 25 15 96 86 1460.7 SDIDEIVLVGGSTR 26 16 86 84  758.4NTVVPTK 27 17 81 1528.7 AKFEELNM(+16)DLFR 28 18 95 1552.8TFAPEEISAM(+16)VLTK 29 19 96 1566.8 ITPSYVAFTPEGER 30 20 96 1604.8TKPYIQVDIGGGQTK 31 21 93 1659.9 IINEPTAAAIAYGLDK 32 22 97 1677.8NQLTSNPENTVFDAK 33 23 99 1836.9 SQIFSTASDNQPTVTIK 34 24 89 1888VTHAVVTVPAYFNDAQR 35 25 91 1934 DNHLLGTFDLTGIPPAPR 36 26 97 2165IEIESFYEGEDFSETLTR 37 27 99 2176 LYGSAGPPPTGEEDTAEKD 38 EL

To further confirm that indeed the ADOPep1 receptor on endothelial cellsis the GRP78 protein, the IP experiment was repeated and Western blotanalysis was performed using an anti GRP78 antibody. Results presentedin FIG. 12 demonstrate the positive staining with anti-GRP78.

Identification of GRP78 Receptor protein on EC under hypoxia—In order toanalyze the receptor on EC that binds the ADOPep peptides under hypoxiaconditions, EC were pre incubated for 5 hours under hypoxia conditionsand immune precipitation was performed with Biotinylated ADOPep1,followed by Western blot analyses using biotinylated ADOPep1 (FIG. 13 a)or anti-GRP78 antibody (FIG. 13 b). Confirmation of an identicalreceptor on EC (glucose-regulated protein, homo sapiens, Gi 6900104)under hypoxia was done by mass spectroscopy (19 identities) as presentedin Table 5, hereinbelow.

TABLE 5 IP with ADOPep1 at Hypoxia IP with ADOPep1 SEQ > Scoreat Hypoxia ID > Mass Sequence NO:  1 96 1588.8 KSDIDEIVLVGGSTR 39  2 961191.6 VYEGERPLTK 40  3 96 88 1217.6 DAGTIAGLNVMR 41  4 92 92 1228.6VEIIANDQGNR 42  5 95 86 1233.6 DAGTIAGLNVM(+16)R 43  6 95 1888VTHAVVTVPAYFNDAQR 44  7 79 1934 DNHLLGTFDLTGIPPAPR 45  8 97 1313.6FEELNMDLFR 46  9 99 1316.6 NELESYAYSLK 47 10 94 1974.9 IEWLESHQDADIEDFK48 11 92 83 1329.6 FEELNM(+16)DLFR 49 12 95 1397.8 ELEEIVQPIISK 50 13 991430.7 TWNDPSVQQDIK 51 14 96 86 1460.7 SDIDEIVLVGGSTR 52 15 95 1552.8TFAPEEISAM(+16)VLTK 53 16 96 1566.8 ITPSYVAFTPEGER 54 17 90 1588.8KSDIDEIVLVGGSTR 55 18 93 1659.9 IINEPTAAAIAYGLDK 56 19 97 1677.8NQLTSNPENTVFDAK 57 20 99 1836.9 SQIFSTASDNQPTVTIK 58 21 97 2165IEIESFYEGEDFSETLTR 59 22 99 2176 LYGSAGPPPTGEEDTAEKDEL 60

Increased presentation of GRP78 protein on EC under hypoxia—FIGS. 14 and15 demonstrate by FACS analysis the percentage of EC expressing GRP78 ontheir membrane under normoxia and hypoxia conditions. As can be seen inFIG. 14, the binding of anti-GRP78 to EC, originating from 10 differentumbilical cords, was 30±13% under normoxia conditions and 52.8±8.4%after 5 hours of hypoxia. FACS histogram (FIG. 15) demonstrated presenceof GRP78 receptor on ECs under normoxia or hypoxia conditions, withincreased binding of anti-GRP78 antibody under hypoxia.

Receptor presence on tumor cells—To further confirm the presence ofGRP78 on EC and its relationship to hypoxia, FACS analyses using theanti-GRP78 antibody performed on different lines of tumor cellsincluding the MCF7 breast carcinoma, SK melanoma, HT colon carcinoma andK562 erythroleukemia tumor cells revealed that GRP78 is expressed onMCF7 breast carcinoma, HT-29 colon carcinoma and SK-28 melanoma celllines but not on K562 erythroleukemia cells (FIGS. 16 a-d).

These results demonstrate that the ADOPeps receptor on EC is GRP78 andthat its presentation on EC membrane is increased under hypoxia and invarious tumor cells.

Example 4 ADOPeps Inhibit Hypdxia-Induced Apoptosis

Experimental Results

Inhibition of the GRP78 receptor with an anti-GRP78 antibody or theaddition of ADOPep1 result in inhibition of hypoxia-induced apoptosis ofEC—The role of GRP78 in apoptosis was studied using EC under hypoxia. Asshown in FIG. 17, the percentage of apoptosis of EC that were exposedfor 24 hours to hypoxia was increased from 25% to 62%. Incubation of ECwith ADOPep1 prevented apoptosis to levels which are similar to thoseseen under normoxic conditions. Incubation of EC with anti-GRP78antibody also decreased the levels of apoptosis, however, the ADOPep wasmore efficient (in 29%) in decreasing apoptosis as compared to theanti-GRP78 antibody (FIG. 17). FIGS. 18 a-e depict illustration by dotplot FACS analysis of the inhibition of apoptosis by the ADOPep1peptide. As can be seen, EC under hypoxia were stained with both AnnexinV and Propidium Iodide (PI) apoptotic markers. In contrast, incubationof EC with the ADOPep1 peptide induced a remarkable decrease in thepercentage of stained cells.

ADOPep1 inhibited hypoxia-induced, but not CoCl₂-induced apoptosis—Tofurther substantiate the effect of ADOPep1 on apoptosis, apoptosis wasinduced by hypoxia or CoCl₂ (Cobalt-Chloride) treatment. As is shown inFIGS. 33 a-f and 34, 24 hours of hypoxia resulted in an increase ofapoptosis to about 70%. In addition, while AdoPep1 inhibitedhypoxia-induced apoptosis of endothelial cells in approximately 40percent, AdoPep1 did not inhibit apoptosis of endothelial cells exposedto the apoptotic inducer Cobalt-Chloride. Thus, the inhibition ofapoptosis by ADOPep1 is specific to the hypoxia stress conditions.

ADOPep1 induces inhibition of hypoxia-induced apoptosis in vivo—As canbe seen in FIG. 22, the mean number of apoptotic cells is dramaticallydecreased in 7-day ischemic hind limb that was injected with ADoPep1.Thus, using the ischemic hind limb mouse model the present inventorswere able to show, for the first time, that administration of ADOPep1 toischemic hind limb results in a significant reduction ofischemia-induced apoptosis.

Altogether, these results demonstrate that similarly to GRP78, astress-responsive protein, the ADOPeps of the invention are capable ofdecreasing the number of hypoxia-induced apoptotic endothelial cells,and therefore can be used to inhibit apoptosis in cells.

Example 5 ADOPeps Bind to the GRP78 Receptor on Endothelial Cells

Experimental Results

Competitive binding of ADAM15 derived peptides ADOPep1, ADOPep2 andADOPep3 to GRP78 receptor—To further test if the ADOPeps bind to theGRP78 receptor on EC, a competitive binding assay was performed on ECwhich were incubated with the ADOPeps prior to binding with theanti-GRP78 antibody. As demonstrated in FIG. 19 (which represents asummary of 4 experiments), all 3 ADOPeps (i.e., ADOPep1, ADOPep2 andADOPep3) show some degree of inhibition of binding of anti-GRP78antibody to EC while scrambled peptide (sRoY) was not effective in thecompetitive binding to the receptor on EC.

ADOPep1 induces upregulation of GRP78 receptor expression in vitro(under hypoxia)—FIGS. 20 a-b further depict the binding of theanti-GRP78 antibody to endothelial cells following incubation of thecells with ADOPep1 under normoxia or hypoxia conditions and demonstratethat while the presence of the GRP78 receptor on endothelial cell (asevidenced by the binding of anti-GRP78 to EC) increases under hypoxiafrom about 18.1% to about 40.1% (as was previously reported by others LiJ, Lee A S. Stress induction of GRP/BIP and its role in cancer. Curr.Mol. Med. 2006; 6:45-54; Arap M A, Landenranta J, Mintz P J, Hajitou A,Sarkis A S, Arap W, Pasqualini R. Cell surface expression of the stressresponse chaperone GRP78 enables tumor targeting by circulating ligands.Cancer Cell. 2004; 6:275-84), a more significant increase in GRP78presentation on the EC (of up to about 83.8%) is observed when the cellsare incubated with the ADOPep1 under hypoxia conditions. These resultsdemonstrate the involvement of ADOPep1 in the upregulation of GRP78under hypoxia.

ADOPep1 induces upregulation of GRP78 receptor expression in vivo (underischemia)—To further confirm the ADOPeps involvement in GRP78presentation on endothelial cells, the mean number of GRP78 positivecells was determined in ischemic limb sections. As is shown in FIG. 21,7 days following induction of ischemia the mean number of GRP78 positivecells was increased as compared to untreated hind limbs. However,injection of Adopep1 resulted in a more significant increase in meannumber of GRP78 positive cells as detected 14 days after induction ofischemia. The relatively low level of GRP78 positive cells at 21 daysafter ischemia probably represents recovery of the ischemia in thetreated animas.

These results demonstrate that ADOPep1 increases GRP78 expression underhypoxia (in vitro) or ischemia (in vivo).

Example 6 ADOPep1 Binding Causes a Grp78 Receptor InternalizationResponse Under Hypoxic Conditions

Experimental Results

ADOPep1 binding causes a GRP78 receptor internalization response underhypoxic conditions—As is shown in FIG. 29, after 5 minutes ofincubation, AdoPep1 induced GRP78 receptor internalization inendothelial cells under hypoxic conditions. The internalization responsewas demonstrated by inhibition of percent binding of AdoPep to membranes(less membrane GRP78 receptors) and increase in intracellular GRP78 meanfluorescence in the endothelial cells incubated for 5 minutes withAdoPep1.

Example 7 Identification of Minimal Motif Sequences from the ADOPepPeptides which Exhibit a Biological Activity

Experimental Results

Identification of novel angiogenesis or tumor related motif—The presentinventors have identified a 4 amino-acid sequence, HWRR, as a commonmotif present on ADOPep1, 2 and 3 ADAM15 derived peptides, that inducesangiogenesis and binds to the GRP78 receptor on EC (Table 6,hereinbelow).

TABLE 6 Amino acid sequences of the ADOPeptides  of the inventionPeptide SEQ ID number Name Sequence NO: 1 ADOPep1

 A H L L P R L P 2 2 ADOPep2 E N F L 

 A H L L 3 3 ADOPep3 A V T L E N F L 

4 Table 6: The bolded text in each peptide amino acid sequencecorresponds to the common 4-amino acid motif HWRR (SEQ ID NO: 5).

The common motif HWRR (SEQ ID NO:5) of all ADOPeps was tested for itsangiogenic activity.

To further test if the 5 amino acid motifs HWRRP (motif A; SEQ ID NO:7),HWRRA (motif B; SEQ ID NO:8) or AHLLP (motif C; SEQ ID NO:6) can bind tothe GRP78 on EC, synthetic peptide having amino acid sequencescorresponding to SEQ ID NOs:7, 8 and 9 were used in a competitive assayfor the binding of biotinylated ADOPep1 to endothelial cells. As shownin FIG. 23, while the peptides having amino acid sequence correspondingto Motif A and B exhibited a significant inhibition of the binding ofADOPep1^(Biot) to endothelial cells, the peptide corresponding to MotifC exhibited a moderate inhibition of binding.

In addition, endothelial cells were incubated under hypoxia conditionsin the absence or presence of ADOPep1, Motif A or C and the Q1level ofapoptosis was determined using FACS analysis. As shown in FIGS. 24 a-dand 25 while ADOPep1 and Motif A and B peptides were capable ofinhibiting the hypoxia induced apoptosis, the motif C peptide exhibitedno effect on hypoxia induced apoptosis.

To further test the biological activity of motifs A, B, C on endothelialcells, a migration assay was performed on EC under hypoxia. As shown inFIGS. 26 a-b, while the ADOPep1, Motif A and B peptides inducedendothelial cell migration at a concentration of about 10 ng/ml, motif Cpeptide exhibited no significant effect on endothelial cell migration.

Altogether, these results demonstrate the identification of minimalamino acid sequences from the ADOPeps which are capable of decreasinghypoxia induced apoptosis and inducing endothelial cell migration. Thesepeptides (e.g., SEQ ID NOs:7 and 8) can be used to inhibit hypoxiainduced apoptosis, induce angiogenesis and treat ischemic diseases.

Example 8 The ADOPeps Motifs are Capable of Tube Formation and Inductionof ERK1/2 Phosphorylation

Experimental Results

ADOPep1 and motif A peptides are capable of forming tubes fromendothelial cells—The ability of ADOPep1 or motif A peptides to formtubes was determined in vitro. As shown in FIG. 27, while the ADOPep1and Motif A significantly increased the length of the network ofconnected cells in endothelial cells under starvation and normoxicconditions, the addition of the scrambled sROY peptide had no effect ontube formation.

AdoPep1 and Motif A peptides compete on the binding to the same receptoron endothelial cells—As shown in FIGS. 28 a-d, AdoPep1 and Motif Apeptides inhibited anti GRP78 binding to endothelial cells under hypoxicconditions. Ten micrograms of AdoPep1 and Motif A inhibited anti GRP78binding in approximately 80% and 60% respectively, while motif C did notinhibit anti GRP78 binding to endothelial cells.

Incubation of endothelial cells with ADOPep1 and Motif A under hypoxiaconditions increase ERK1/2 phosphorylation—As is shown in FIGS. 30 a-b,incubation of endothelial cells with ADOPep1 and Motif A under hypoxiaconditions resulted in a significant increase in ERK1/2 phosphorylationas measured after 20 minutes.

Induction of ERK1/2 phosphorylation by ADOPep1 and motif A peptides isspecific—In order to assess that ERK phosphorylation is specific toAdoPep1 and Motif A activation, a specific pERK peptide inhibitor wasadded to the endothelial cells incubated with AdoPep1 and Motif Apeptides. FIG. 31 shows Western blot analysis of ERK phosphorylationinhibition by the inhibitor peptide in endothelial cells incubated withAdoPep1 and Motif A for 20 minutes under hypoxic conditions.

ADOPep1 and ADOPep2, but not ADOPep3 are capable of inducing ERK1/2phosphorylation in endothelial cells under hypoxia—As shown in FIGS. 32a-b, Western Blot analyses performed using anti Phospho ERK antibody(Santa Cruz Biothechnologies p-ERK(E-4),sc7383) revealed that whileADOPep1 and 2 induced ERK1/2 phosphorylation, ADOPep3 had not effect onERK1/2 phosphorylation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the invention.

REFERENCES Additional References are Cited in Text

-   1. Trochon V., Li H., Vasse M., Frankenne F., Thomaidis A., Soria    J., Lu. H., Gardner C. and Soria C. Endothelial    metalloprototease-disintegrin protein (ADAM) is implicated in    angiogenesis in vitro. Angiogenesis 2: 277-285, 1998;-   2. PCT Pub. WO2005/039616 to the present inventors-   3. Koomaqi R., et al., 1999; Anticancer Res. 19(5B): 4333-6;-   4. Dong D., et al., 2005, Cancer Res. 65(13): 5785-91;-   5. Koshikawa N., et al., 2006, Oncogene 25(6):917-28;-   6. Davidson D J., et al., 2005, Cancer Res. 65(11): 4663-72;

1. An isolated peptide consisting of the amino acid sequence HWRRP setforth in SEQ ID NO:7 or HWRRA set forth in SEQ ID NO:8.
 2. (canceled) 3.An isolated peptide comprising an amino acid sequence HWRRP as set forthby SEQ ID NO:7 or HWRRA as set forth in SEQ ID NO:8, wherein the peptideconsists of 12 or less amino acids.
 4. The isolated peptide of claim 3,wherein said amino acid sequence is set forth by SEQ ID NO:2 or
 3. 5-6.(canceled)
 7. The isolated peptide of claim 3, wherein the peptide is alinear peptide.
 8. The isolated peptide of claim 3, wherein the peptideis a cyclic peptide.
 9. The isolated peptide of claim 3, wherein thepeptide consists of 12 or less amino acids.
 10. A composition-of-mattercomprising at least one peptide of claim
 3. 11. A pharmaceuticalcomposition comprising as an active ingredient at least one peptide ofclaim 3 and a pharmaceutically acceptable carrier or diluent.
 12. Amethod of inducing angiogenesis in a subject, the method comprisingadministering to the subject a therapeutically effective amount of atleast one peptide of claim 3, to thereby induce angiogenesis in thesubject. 13-14. (canceled)
 15. A method of treating a pathologycharacterized by insufficient angiogenesis in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of at least one peptide of claim 3, to thereby treat thepathology characterized by insufficient angiogenesis in the subject. 16.(canceled)
 17. The method of claim 15, wherein the pathologycharacterized by insufficient angiogenesis in the subject is selectedfrom the group consisting of delayed wound-healing, delayed ulcerhealing, reproduction associated disorder, arteriosclerosis, ischemicvascular disease, ischemic heart disease, myocardial ischemia,myocardial infarction, heart failure, myocardial dysfunction, myocardialremodeling, cardiomyopathies, coronary artery disease (CAD),atherosclerotic cardiovascular disease, left main coronary arterydisease, arterial occlusive disease, peripheral ischemia, peripheralvascular disease, vascular disease of the kidney, peripheral arterialdisease, limb ischemia, critical leg ischemia, lower extremity ischemia,cerebral ischemia, cerebro vascular disease, retinopathy, retinalrepair, remodeling disorder, von Hippel-Lindau syndrome, diabetes,hereditary hemorrhagic telengiectasia, ischemic vascular disease,Buerger's disease and ischemia associated with neurodegenerative diseasesuch as Parkinson's and Alzheimer's disease.
 18. (canceled)
 19. Theisolated peptide of claim 3, wherein said peptide binds aglucose-regulated protein (GRP78) as set forth by SEQ ID NO:9 onendothelial cells of the tissue.
 20. (canceled)
 21. A compositioncomprising an agent attached to the peptide of claim 3, wherein thepeptide targets said agent to endothelial cells.
 22. The composition ofclaim 21, wherein the agent is selected from the group consisting of atoxin, a chemotherapeutic agent and a radioisotope.
 23. A pharmaceuticalcomposition comprising as an active ingredient the composition of claim21 and a pharmaceutically acceptable carrier or diluent.
 24. A method oftreating a pathology characterized by abnormally increased angiogenesis,comprising administering to a subject in need thereof a therapeuticallyeffective amount of the composition of claim 21, thereby treating thepathology characterized by the abnormally increased angiogenesis. 25.(canceled)
 26. The method of claim 24, wherein said pathology isselected from the group consisting of cancer, metastatic cancer,myelodysplasia, Systemic mastocytosis (SM), retinal neovascularization,neovascularization in atherosclerotic plaques, hemangiomas, arthritis,and psoriasis.
 27. (canceled)
 28. A method of identifying a putativeangiogenic molecule, the method comprising: (a) incubating the peptideof claim 3, with a glucose-regulated protein (GRP78) or cells expressingsaid GRP78 under conditions suitable for formation of a complex betweenthe peptide and said GRP78 or said cells expressing GRP78, and (b)identifying a molecule capable of displacing the peptide from saidcomplex, to thereby identify a putative angiogenic molecule.
 29. Apharmaceutical composition comprising as an active ingredient at leastone peptide of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 30. A method of inducing angiogenesis in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of at least one peptide of claim 1 to thereby induce angiogenesisin the subject.
 31. A pharmaceutical composition comprising as an activeingredient at least one peptide of claim 4 and a pharmaceuticallyacceptable carrier or diluent.
 32. A method of inducing angiogenesis ina subject, the method comprising administering to the subject atherapeutically effective amount of at least one peptide of claim 4 tothereby induce angiogenesis in the subject.
 33. A composition comprisingan agent attached to the peptide of claim 1, wherein the peptide targetssaid agent to endothelial cells.
 34. An isolated peptide consisting ofthe amino acid sequence set forth in SEQ ID NO:4.
 35. A pharmaceuticalcomposition comprising as an active ingredient the peptide of claim 34and a pharmaceutically acceptable carrier or diluent.
 36. A method ofinducing angiogenesis in a subject, the method comprising administeringto the subject a therapeutically effective amount of the peptide ofclaim 34, to thereby induce angiogenesis in the subject.